Continuity mistake: In the scene where the lizard and Spiderman are fighting in the sewers, the lizard scratches Spider-Man's chest, causing tears down the front of his suit and chest. He actually scratches straight down his chest, but from then on, whenever Spiderman's chest is shown the scratches are diagonally across him.
Continuity mistake: Inside the school lab, the Lizard walks to Peter on the floor and a stool next to him suddenly appears for him to pick up. (01:35:00)
Continuity mistake: In the scene where Peter Parker is fighting on a train he tells the last man standing 'not my board', the last man is black but after hitting Parker and the board breaking the next shot is one of the guys he has already knocked down who is white fighting him after the board breaks.
Continuity mistake: When Gwen visits Peter at the end of the movie, you can see that the glass door with the house number "36" painted on it has been replaced from when he broke it earlier in the movie, and it looks brand new. Once she leaves and he turns to Aunt May, the numbers have returned to their earlier weathered and peeling state.
Factual error: There is a scene in midtown Manhattan toward the middle of the film outside a NYC subway station, which according to the sign on the subway entrance is at 6th Avenue and 39th Street. There is no such subway station on those streets.
Continuity mistake: When Peter is looking through his father's file in his bedroom, Uncle Ben comes in and starts playing with a Rubik's cube from Peter's desk. When he sets it down, the top and middle row are red and the bottom row is white. Shortly afterwards, when Peter is researching Doctor Connors on his computer, the top and middle rows are suddenly white and the bottom row is red (not fully solved, as another listed mistake claims).
Continuity mistake: During the scene where Uncle Ben is talking to Peter in Peter's bedroom, on his desk there is a solved Rubik's cube. Uncle Ben picks it up, and twists it slightly. It then suddenly appears on the opposite side of the desk next to Peter, too far away for Ben to have placed it there, fully solved again.
Continuity mistake: When the one of the cups of coffee falls off the table Peter catches it. After that, Peter takes the other cup from the desk to give Dr. Connors. In the first shot Peter carries the cup from the upper part, next shot the cup is on his palm.