(at around 24 mins) In the bus fight scene when Peter's powers are starting to kick in, an African American male hits him with his own skateboard, but then immediately turns into a white guy the next shot.
(at around 33 mins) When Flash hits the girl's can of paint with the basketball in the gym, a lot of paint is spilled but when Peter lifts up the can only a little bit of paint is spilled.
(at around 1h 27 mins) When Lizard finds Spider-Man in the sewer on his web (when trying to get pictures of Lizard), Lizard scratches Peter from the top of his chest down. In the next scene where Peter shows up at Gwen's window, his cuts go from the top of his left shoulder diagonally down to the right side.
(at around 1h 12 mins) When Peter leaves Gwen's apartment by jumping off the balcony, you can see he forgot his backpack. In the next scene however, he is carrying it again.
(at around 49 mins) The first time that Peter attacks a guy in the city, while he is being chased the men climb to the roof much too quickly; by the time Peter gets to the top they are arriving at the roof. In reality, it would take much longer to climb those staircases.
(at around 1h 45 mins) Towards the end of the movie Gwen Stacy sets off the fire control system and hides in the Oscorp lab, causing the fire doors to shut and fire fighting agent to be released. The agent was either; CO2, and would fight fires by displacing all of the oxygen in the room resulting in an asphyxiated Gwen, or Halon (commonly used in industrial settings) that can cause nerve damage when in high enough concentration and with prolonged exposure as well as asphyxiation.
(at around 1h 18 mins) While a Police Chief, Captain or Commissioner can issue an order to arrest someone wanted for questioning, they cannot issue an arrest warrant as Captain Stacy did. Only a judge can issue an arrest warrant.
(at around 1h 50 mins) When Captain Stacy is firing his shotgun at the Lizard, he fires way more shells than a shotgun can hold without reloading at all.
(at around 1h 35 mins) While hunting Dr. Connors in the sewer, Peter receives a cell phone call. Cell phone signals don't reach underground areas without a picocell.
Dr. Connors' right arm-stump is very muscular. However, as anyone who has worked with amputees knows, after several years of having an amputated limb, the remaining stump still attached, experiences degeneration and is withered.
(at around 1h 23 mins) When Peter describes Doctor Connors to Captain Stacy, Peter grabs his left arm to indicate the missing arm. Doctor Connors is missing his right arm. This is not an error but a common thing. Functionally, it is as if one is looking into a mirror. From Peter's perspective, Connor's missing limb is to his left and his automatic reaction is to indicate his own left arm.
(at around 1h 45 mins) Spider-Man falls violently onto a fire escape which is clearly made of rubber as it can be seen bending and vibrating.
(at around 5 mins) When Peter is taking a picture of Gwen, there is a point of view shot of Peter's rangefinder camera that shows the viewfinder markings not consistent with rangefinder cameras. It shows instead an SLR camera's viewfinder markings, something people are more familiar with.
At around 1hr 35, in the lab fight scene, Peter gets thrown through a wall. There's a lot of shake and the wall partially collapses, creating a hole. None of the glassware on the adjacent shelving shakes, wobbles, rattles, falls over or breaks. Likewise, a few seconds later when a beaker of chemicals is thrown through the hole, there's an explosion causing the room to shake again, but none of the adjacent glassware moves even slightly.
During the fight in the library Stan Lee exits just as Spider-Man is thrown behind him. This CG Spider-Man is smaller than he should be.
The Lizard finds out Spider-Man is Peter Parker when he sees the label at the back of one of the cameras reads "Property of Peter Parker". It is because that was a 'hole' in Peter's planning, it's not like he wanted a villain to find out his secret identity.
(at around 1h 50 mins) Towards the end of the film, Gwen leaves the Oscorp tower to meet Captain Stacy at the sidewalk. We hear the sound effect of his car screeching to a stop, but the car is already stopped with the door open from the start of the scene.
When Dr. Connors is talking to Norman Osborn's representative, and when Capt. Stacy is talking to Peter in the station, we see shots of both these characters from the side. Their lips do not match up with the audio.
(at around 30 mins) When Dr. Connors and Peter are talking in his house, you can see a green screen in Dr. Connors glasses.
While Peter is talking with Dr. Connors in doc's house you can clearly see the optic of the camera on the top right of the window right behind Dr. Connors.
(at around 23 mins) When Peter first discovers his powers on the train (Q-train), it was leaving Manhattan (Oscorp Tower) heading towards Coney Island, Brooklyn (last stop). Peter Parker lives in Forest Hills, Queens, NY. The Q-train did travel to Queens at the time of filming, but it went to Astoria at the north-western tip of the borough. The correct trains for Peter to be on would be the R M F or E. Additionally, the Q ran above ground in most of Brooklyn, including the approach to Coney Island.
(at around 14 mins) In an establishing shot of the location of the Oscorp tower, when Peter first visits, it is actually the Hearst building located on the southwest corner of 8th and 57th. Then in a later aerial establishing shot they show it to be behind Grand Central Station, some 20 blocks away in the current location of the MetLife building.
(at around 39 mins) When Dr. Connors says some lizards can regrow lost limbs "at will". The regrowth is an automatic process like hair growth on mammals, no "will" is involved. He is imputing will to a natural process, a rather clumsy anthropomorphic metaphor for someone who should know much better.
(at around 1h 40 mins) When the police SWAT team are seen approaching Spider-Man after he had been downed by the stun dart, one of the officers is looking through the scope of his automatic rifle with the flip cover closed, meaning he wouldn't be able to see through the scope.
(at around 29 mins) When Peter is reading on the house roof, reading Dr. Connors' book, we see a spelling mistake in the book. Instead of; "Where do we go from here..." It is spelled as "Were do we go from here..."
(at around 16 mins) Curtis Connors explains that herpetology is "the study of reptiles." It is actually the study of both reptiles and amphibians. Such an omission is particularly odd for his character.
(at around 1h 50 mins) At the climax of the film, the countdown for the launch of the projectile begins with "Detonation in T-minus two minutes". "T minus ..." indicates something is going to happen in a set time. The announcement should have either said "Countdown at T minus 2" or "Launching in 2 minutes".