When the Mario Bros. are in the elevator with the dancing Goombas, they are wearing their specialized jump boots. When they get out of the elevator, they are wearing their normal shoes.
When Lena steps on the Rock to keep it from falling through the grate, the length of cord that has fallen through the grate changes between shots.
In Devo 4 Luigi's button unbuttons and rebuttons itself.
When the van breaks down at the beginning of the film, it has some lengths of piping strapped on the side and overhanging out a couple of feet at the back. Once the van has stopped, the copper piping is nowhere to be seen.
While on the ledge just before entering the alternate dimension, the Mario brothers switch places.
On the Blu-ray edition, during the sequence in which the Bob-omb is crossing the road (at around 1h 30 mins), the string pulling it across can be seen clearly.
Flipped shot. After the brothers steal a police car, the text on the side is momentarily reversed.
At the ledge just before they enter the alternate dimension, Mario says "Watch it!" as he and Luigi switch places, but his lips don't move.
How did the Mario brothers not recognize King Kooper when his face is on billboards and posters all over dinohattan.
When Spike asks Iggy the square root of 26481, he answers 191, which is the square root of 36481.
When Mario and Luigi steal a police car and are being chased, as they enter the tunnel there is a message on the computer screen that says "WARNING: TUNNEL NOT FINNISHED", which doubles the N in "finished".