Other mistake: Near the beginning of the movie, Milla Jovovich reads a note that has been left on the desk. She didn't write it, as they demonstrate in the film. However, when they do show the person who wrote the note composing it, the handwriting is different from that in the note from the earlier scene.
Continuity mistake: When Matt gets scratched by the mutated Licker at the end of the film, the scratches on his arm appear before the creature's claws actually hit him.
Factual error: When the special forces arrive in the Station of the hive, the computer identifies the soldier`s weapons. The name under the picture is right, but the picture always shows a MP5, even at the soldier carrying a G36 Assault rifle.
Plot hole: Red Queen is extremely conveniently programmed for the plot of the movie. 1. Rather than seal off rooms in a place she has TOTAL control of, (that by the way is designed to be isolated in sections for this very reason), she lets the infection spread to everybody in the Hive. She could have easily contained it in a small section of the facility. This is standard procedure in ANY biowarfare lab, there is no way she wouldn't be programmed to do it. 2. Rather than let everyone know the virus is loose, she just starts killing everyone. She had counter-virus available, why not let everyone know that they are locked in with the virus, but help is coming? The infected personnel would be hit with it only minutes after infection. If it didn't work and they died, they are still locked in. 3. Instead of calling Umbrella and telling them the virus is loose, she clams up and just slaughters everyone. This guarantees that Umbrella will ignore anything she has to say, and breach the Hive with a response team and release the virus, the very thing she is programmed to prevent. 4. When Kaplan is disabling her, she keeps telling him that it's a bad idea, but doesn't bother to let him know that shutting her down will release hordes of flesh-eating killers. 5. She just assumes that Spence, no matter how he got in to the T-virus storage has clearance to stuff it all in a duffel bag and walk out with it. She could have locked him in and double-checked with Umbrella. 6. The entire reason for her existence is to prevent the virus from escaping, while almost every decision she makes greatly increases the chances it will get out of the Hive. This is way beyond bad programming - this is highly advanced AI doing the exact opposite of what she's designed for.
Revealing mistake: When the group is being attacked in dining hall B, they are trying to get the doors open. J.D. is firing at two zombies and turns away to open the doors himself because Kaplan keeps forgetting the code. If you look closely, one of the zombies reacts a second late from being shot down.
Factual error: In the laser corridor, when the grid of lasers is formed, some of the lasers rotate to form the grid, and are no longer emitted from anything, they are just hanging in the air.
Continuity mistake: Near the end of the film, on the train ride out of the Hive, Rain is quite close to becoming undead. When she speaks, we notice her teeth are still "movie-star white". However, a few shots later, when she becomes a zombie, she sort of hisses where you can see it has taken what couldn't have been more than a few mere minutes for her teeth to become brown and discolored.
Revealing mistake: When the team leader is opening the door to the Red Queen chamber, he clips an electronic device to the door to hack the code. The device is actually a blood glucose monitor as used by diabetics - an Accu-Chek, I think.
Other mistake: When the train arrives back under the mansion from the Hive, it enters the station from the wrong direction. Looking at the track from the platform, the train comes in from right to left, when it should be from left to right.
Revealing mistake: On the DVD commentary, the director says that he only had a handful of the experiment cubes in Dining Hall B and used cardboard cutouts for all the others. This is most obvious when Rain is walking through the hall alone to check on the first zombie: the cubes in the distance don't match the rigid rows of the ones in the foreground.
Other mistake: Watch Milla Jojovich's hair during the scene when they are held at gunpoint by Spence. Her hair moves from shot to shot. For the most obvious, watch right after Spence shoots the zombie.
Revealing mistake: Near the end right before the end credits we see the streets of Raccoon City completely destroyed, but if you look carefully you can see the background is repeating itself street by street. The best way to notice this is to look at the firetruck right before the end. There is a firetruck, a news van and a bus. Now look to the next street on the other side of the road and the exact same cars appear there.
Continuity mistake: When Alice escapes the first zombie dog and locks herself in the room, a zombie approaches her and she kicks him down. When the zombie falls you can see his tie is partially outside of his suit. When it cuts to Alice reaching for his gun, the tie is tucked inside his suit.
Plot hole: Why didn't Umbrella's higher ups just ask Red Queen what happened before sending in an assault team? She could have told them the T-virus was released and the hive would have stayed sealed. Instead, they just assumed she was nuts and sent in people to shut her down! Had even one of them said, "What happened?", the zombies would have never been released - it's a plot hammer that contradicts the whole point of having an AI run the hive. There should have been a line that said something like "All attempts to communicate with Red Queen so far have failed. The whole point of an AI that has the capability to seal the Hive is to keep the virus from escaping! Her refusal to communcate guarantees that the hive will be breached by a team that is investigating.
Visible crew/equipment: When the team leader asks Alice for her report, if you look in the reflection of the eye pieces on his mask, next to Alice's head, you can see a man wearing jeans. This happens twice.
Audio problem: When Spence is killed by the Licker, Matt sees this on the screen and asks, "What the f**k was that?" Where did he find the time to practice his ventriloquism? You can see that his jaw isn't moving...