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: 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.
Continuity mistake: During The Lizard's attack on the bridge, he throws the car with Doctor Ratha in off a bridge. Peter then catches the car with a web. Yet as soon as he notices The Lizard, not only is the web not in his hand anymore, but there's no sign of it hanging over the bridge either. Then the web and car reappear in the wide shot where Peter swings over to get the boy out of the car.
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.