(at around 20 mins) When Optimus Prime arrives at Autobot City and begins wiping out Decepticons, he is seen shooting Soundwave. However, Soundwave is clearly seen later on unhurt, carrying Megatron's damaged body.
(at around 10 mins) Sunstreaker is seen doing road construction early on in the movie, he is present amongst the Autobots setting up the roadblock for Kup. He is also seen next to Kup when he sees Hot Rod shooting at the shuttle. Yet later in the movie, he is seen piloting the shuttle that Optimus Prime has taken to arrive to Earth, and is standing next to him as he transforms to go face Megatron.
(at around 1h 16 mins) When Hot Rod successfully opens the Matrix, he is able to fit his fingers into square holes in the handles. These finger holes do appear in a couple of other scenes (when Galvatron claims the Matrix from Magnus, and when Galvatron tries to claw it open to use against Unicron), but they seem to be an afterthought. The finger holes are missing throughout most of the movie, including the scene in which Optimus Prime first takes the Matrix out of his chest.
(at around 17 mins) When the Constructicons form Devastor for the first time, his chest plate is the same color as the rest of his body but then switches back to its regular color, purple, in the next shot.
(at around 7 mins) We watch Prowl die with flames beginning to shoot out of his mouth, and eventually he falls on his back with his whole torso in flames. Later, as the Decepticons take over the controls of the Autobot ship, the remains of the four autobots are seen on the floor, and Prowl is lying face first with much of his torso still intact. In fact, very little damage is shown on all four fallen Autobots until Ironhide reaches for Megatron in desperation.
(at around 33 mins) When Galvatron interrupts Starscream's coronation Thundercracker and Skywarp and some Insecticons can be seen clearly among the Decepticons there. Thundercracker, Skywarp and the Insecticons were among the damaged Decepticons thrown from Astrotrain with Megatron. Skywarp became Cyclonus, Thundercracker became Scourge and the rest became Scourge's Sweeps.
(at around 28 mins) Astrotrain can apparently change his size to be able to accommodate all the other Decepticons (who are generally about as big as he is) inside himself when he transforms into his alternate modes. This strange phenomenon is called "mass shifting", but one particular scene takes it to the extreme: The Constructicons merge to form the gigantic Devastator, inside Astrotrain. This is considered to be an error, since for the Constructicons to be able to unite, Astrotrain would have to grow immensely huge, bigger than even the inconsistent senses of the Transformers cartoon would allow.
(at around 2 mins) There are a couple frames where you can see the inside of one of the Lithonian ships being devoured by Unicron. The pilot of the ship appears to be Kranix, even though he apparently screams out Kranix's name, and Kranix appears later in the Quintesson prison.
When the Decepticons attack the Autobot ship in the beginning. The Decepticons blow a hole on the side of ship and enter to begin the attack. In reality if they did this, the Vacuum of Space would of immediately sucked everything out into space.
(at around 1h 19 mins) The voice actor credits include listings for the characters Inferno, Gears, Prowl, and Dirge, even though none of them had speaking lines in the movie. Also, Jack Angel does not receive a credit for the character of Ramjet, even though Ramjet's voice can clearly be heard when the injured Decepticons are pushed off of Astrotrain and he commands, "Get! Make room for others!" For that matter, Frank Welker doesn't get a credit for his portrayal of the auto-combatant that Hot Rod is sparring with. The cast list is supposed to be alphabetical, but Peter Cullen's name is listed before that of Scatman Crothers.
(at around 25 mins) When Ultra Magnus is passed the Matrix of Leadership from Optimus Prime, he brings it to his chest, inserts it and then re-inserts it to adjust its placement. This shows that Ultra Magnus is not built to correctly keep the Matrix, and thus not meant to be the Autobots' true leader.
(at around 1h 14 mins) When Daniel knocks down half of the acid vat's cover and Bumblebee, Jazz, Cliffjumper, and Spike land on it, none of Unicron's other victims follow them, even though they weren't the last to be eaten.
(at around 1 min) As Unicron is about to devour the first planet at the start of the film, there's a shot of a robot looking up towards him saying "it's Unicron!", at this part there's an animation error. If you look carefully the bottom part of Unicron's outer ring overlaps an object that is in the foreground, this would mean that Unicron is actually smaller than the objects on the surface of the planet and not bigger than the planet overall as established by the next few shots.
(at around 50 mins) When the Allicons (alligator-like guards of the Quintessons) come out of the water reacting to the appearance of Hot Rod and Kup, there are splashes around their feet even when they are on land. Later, when they transform to attack the newcomers, you can see dark-colored stripes running down their features. These were supposed to mark the line under which darker tones should be applied to make a shadow effect, but no shadow is painted onto them, only the darker stripes remain visible.
(at around 23 mins) As Soundwave carries the damaged body of Megatron through the battlefield towards Astrotrain, followed closely by Rumble carrying Megatron's cannon, they appear to be running in thin air, as no ground is painted beneath their feet.
(at around 15 mins) When Blaster ejects his tapes, he first ejects what looks like Eject. He stays blue up until he gets to the edge of the screen. He then turns black like Rewind. Then Blaster ejects another blue cassette, which after about a second, turns black. This one gets through half his transformation colored black, then turns blue for a split second then turns black again. Then, when the cassettes are fighting each other, Eject runs in from the left and jumps over Perceptor. And then he runs in from the left again to shoot Ravage.
(at around 1h 18 mins) When Rodimus Prime transforms for the first time and drives off, look at his windshield: the top of it keeps changing shape from frame to frame, creating a weird effect when viewed in motion.
(at around 6 mins) Back on Cybertron, when Soundwave retrieves Laserbeak, he doesn't push the eject button on his shoulder to open or close his cassette door. This is not only inconsistent with previous episodes, but also with scenes later in the movie.
When Starscream carries the wounded Megatron to the airlock to be jettisoned. Soundwave's face visor is coloured grey, like the rest of his face. Fans have often joked that he is turning a blind eye to Megatron's liniment demise.
At 56:45, when Springer, Arcee and Ultra Magnus and Daniel are fighting off an attack by Galvatron and the Sweeps on the Planet of Junk, it's Decepticons Scrapper and Shrapnel who fall out of the way to block Daniel's way. These two Decepticons were earlier destroyed and revived by Unicron as Sweeps.
(at around 22 mins) When Megatron shoots Optimus Prime he shouts "Fall! Fall!". However, the lip syncing on Megatron is out of place as he says it, instead hinting by the animation that he would've originally said at least three words instead of two.
(at around 41 mins) When Hot Rod suggests that Kup has better things to do than tell stories to the Dinobots, the Dinobots begin protesting. One of them says, "Quiet! We wanna hear story!" It looks like Sludge's mouth is moving here, but this definitely isn't Sludge's voice.
(at around 49 mins) The first time we see Wreck-Gar, his mouth doesn't move when he delivers the line, "Offer expires while you wait. Operators are standing by."
(at around 20 mins) After Optimus Prime plows through the group of Decepticons in truck mode, there's no transforming sound effect when he changes back to robot mode.
(at around 27 mins) During the voting process to keep the wounded on board Astrotrain or dump them into space, the voices of the Decepticons chanting "Aye!" and "Nay!" don't match any of the Decepticon characters present.
(at around 1h 1 min) After getting a new spacecraft from Quintessa, Hot Rod, Kup, Wheelie and the Dinobots fly over to the planet of junk to meet up with the other autobots. However, they had crashed on Quintessa earlier whilst the other autobots flew onwards to the planet of junk, and seeing as they hadn't contacted them at all since the crash, how could Hot Rods group possibly know which planet the others were currently on?
(at around 38 mins) After Galvatron says "Ultra Magnus is mine!", thereby implying that he wants to be the one to destroy him, he proceeds to attack Hot Rod, Arcee, and the Dinobots instead of Ultra Magnus.
(at around 6 mins) It is unexplainable how Laserbeak managed to get video recording of the Autobots from all those angles we see when he plays the recording back, considering he was stationary during the recording process. Some of his shots indicate that he moved right in front of the faces of the Autobots, which would have been impossible for him to do, partly because he was outside of their base and partly because the Autobots would have noticed him.
(at around 50 mins) Kup and Hot Ron run out of Energon when they offer them to the Alicons, and they even toss away the empty container. Yet when they arrive on Planet Junk (at around 1h 4 mins), Hot Rod makes piece with the local Junkions by offering them more Energon.
Numerous inconsistencies in how long it takes to travel from Cybertron to Earth. Optimus Prime takes about a day to get there, depending on how long it took him to receive the distress signal. It is the following day when he arrives, and presumably it took Megatron the same to get there in the stolen Autobot shuttle. After the battle, however, they receive the radio signal from Cybertron almost instantaneously. Galvatron, who receives orders from Unicron at the same time, arrives to Earth in a matter of minutes.
(at around 1h 5 mins) When the Junkions start to reassemble Ultra Magnus, they begin by putting his legs on backwards. They try to fit his left leg into the right socket, and vice versa.
(at around 29 mins) Unicron changes sizes many times from being 10 times bigger than the planet he eats in the beginning to when Megatron encounters him for the first time.
(at around 27 mins) Soundwave is the most loyal of Megatron's followers. He carried him back to Astrotrain when all the Decepticons were retreating. However, when Starscream throws Megatron into space, Soundwave just stands aside and allows him to do it.
Soundwave's sudden act of not intervening when Starscream is about to cast the wounded Megatron out in to space. Seems to just come out of nowhere, considering his portrayal of loyalty to Megatron.
Megatron says, "I would've waited an eternity for this," while standing over the fallen Opitmus Prime. Literally, waiting forever means it would never happen.