Plot hole: Why does Umbrella know that Red Queen killed everyone, but not that the virus is loose? Is there no way to see into or receive data from the Hive other than Red Queen? Does every communication in or out have to be approved through her? And even if she is the sole conduit for information into and out of the Hive, her primary purpose for existing is to keep the T-virus from escaping. Umbrella THINKS that Red Queen has malfunctioned. BUT, she is working perfectly! So suppressing info that the T-virus is loose in the Hive would contradict her core programming. So why doesn't Umbrella know?
Deliberate mistake: When the woman is attempting to escape from the elevator, it dislodges and falls down, so then the actress is facing the ground...but when her arm hits the floor, the floor itself bends. If you watch the DVD with the commentary on, the director points out that it was a break away floor in case the actress' head accidentally snapped forward, to avoid any injury. (00:08:00)
Continuity mistake: Shortly after the Licker pulls Kaplan out of the train, Matt shuts the door so it can't get at the rest of them. The Licker goes on the roof to use the back entrance to get into the car. Matt hears this and rushes to bar the back door. Pay attention to the near dead Raine. She suddenly gets a lot more life and quickly moves out of the way to avoid being accidently trampled. (01:24:55)
Continuity mistake: When Matt is at Lisa's desk he looks through some papers, he is looking at a paper which has black squares on it. Then he puts the paper down and looks at another paper. When it cuts to him noticing Lisa approaching he is now holding the paper with the black squares again. (00:50:20)
Revealing mistake: In the scene where Milla Jovovich shoots the dogs with the 9mm Beretta she obtained from the dead security guard, a closeup of the gun is shown. Watch the cycling of the slide and you will notice that the ends of the bullets are crimped, indicating the use of blanks. (00:49:55)
Continuity mistake: When we see the extreme close up of the robotic arm picking up the blue vial, the grip is uniform and parallel to the edge of the sliver end cap on the vial. When we see the vial a second later, the arm's grip has changed and is no longer parallel or uniform on the vial. (00:02:10)