(at around 1h 20 mins) When Jefferson talks to tied-up Miles, he references how he can see his shadow moving; however, when Jefferson checks the shadow a second time, the shadow is the same size, despite Miles being up against the door. You can see that Miles' shadow is not being cast under the door when the camera cuts to show both sides of the door.
(at around 58 mins) When Peter Parker, Miles Morales and Gwen Stacy stand outside Aunt May's door, Peter starts walking away until Gwen fires up a web to pull him back. In the very next shot, Peter is back in place but the web behind him is gone entirely.
(at around 1h 11 mins) In the fight between the Spider-Men and the villains at Aunt May's House, Aunt May knocks Tombstone outside with a bat. Seconds later he can be seen briefly in the background fighting in the house again.
(at around 16 mins) Miles has Gwen's hair stuck to his hand after Gwen's head is shaved to free them. In the next shot, as Miles walks down the hall, his hands are clean. Later, when Miles is confronted by a security guard, the hair is stuck to his hand again.
(at around 1h 29 mins) At the collider fight, Dr. Octopus wraps up Spider-Man with two of her arms, uses one to hang to the ceiling and another to attempt to kill him. In the wide shot when Miles uses her own arm to punch her with it, there're two arms on the ceiling, one wrapping up Peter. When Miles reveals himself. it reverts back to two of the arms wrapped around Peter.
(at around 1 min) Mary Jane of Miles' universe is shown kissing Spider-Man while hanging upside down on a ladder, but her hair stays upright and perfectly styled. Realistically it should be hanging down towards the ground.
When Prowler is shot in the back, he is pushed backwards towards the gun, instead of away.
After Prowler is shot in the back, Kingpin cocks the hammer on his pistol to fire it again. The weapon he's using appears to be a simplified version of the Colt 1911. Its hammer is automatically cocked as the receiver cycles, ejecting the spent shell casing and chambering another.
Miles is nearly a foot shorter than Peter Parker, yet when Miles takes one of Peter's old costumes to use as his own, it fits him perfectly. This was done intentionally by the filmmakers, meant to be an illustration of Miles' earlier conversation with Stan, the comic shop owner ("It always fits. Eventually."). Miles has grown more confident, more skillful, and more comfortable with his new powers and is finally ready to take on the role of Spider-Man; thus the costume (like the role itself) finally "fits".
(at around 1h 12 mins) Miles' dad lives in Brooklyn, where he gives Miles a ride to school, yet when the villains show up at Aunt May's house, which is in Queens, he picks it up on the radio and drives over. It's never said that he is a Brooklyn cop. Nothing prevents him from living in Brooklyn and driving a police car there, while working in Queens, especially since PDNY probably serves all the boroughs like NYPD does.
(at around 6 mins) You famously can't unlock the back door of a police car from the inside, yet Miles gets out of his father's squad car unassisted. It is plausible that this is just one of many difference between our universes. It does make certain sense to give officers the ability to control whether the door can be opened from inside or not, just like in most modern private cars.
Jefferson has what appears to be lieutenant bars on his collar, yet he also has a chevron on his sleeves. If he is a police lieutenant, he would not have chevrons, as this indicates the rank of corporal. This may be one of the alternate universe's many subtle, quirky differences from ours, e.g. PDNY vs. NYPD.
(at around 46 mins) Kingpin is bigger than the SUV he exits at Alchemax, which makes no sense unless it's a TARDIS. It's a joke.
(at around 56 mins) When Gwen says, "You don't get to like my haircut," the lip movement is different.
(at around 1h 19 mins) When the group of Spider-people leave Miles' dorm room after he gets tied up, they all shoot webs upwards and swing from them to get across the city; however, there doesn't appear to be anything for them to attach their webs to.
(at around 57 mins) When Doc Ock confronts Kingpin at Alchemax and holds the henchman with her tentacles, the henchman points his guns at her; however, there is no way he can see past Kingpin's huge frame to shoot her. She is in no danger of being shot.
(at around 36 mins) On a report Peter B. Parker briefly reads in a flashback, a scathing review for a Spider-Man-inspired restaurant named TGI Spideys begins with, "Spider-Man is New York's hero, it's savior," misspelling "its."