Revealing mistake: Right at the end of the fight between Selene and the Uber-Lycan in the vampire coven, the Uber-Lycan throws Selene into some wooden panels, knocking her unconscious. As she travels through the air you can tell that she is suspended by wires; she travels far too slowly towards the wooden panels for someone who was just thrown.
Factual error: Detective Sebastian fires a total of 7 rounds at the Antigen van in the parking garage - three at the driver, two at the front-left tire, one right before the van nearly runs him over and then one after it passes him - without reloading, yet the Taurus Judge revolver he is using is designed to only hold 5 rounds in its cylinder. Later, after Sebastian has reloaded the revolver, he fires 8 rounds at Dr. Lane, again without reloading.
Visible crew/equipment: When Detective Sebastian is talking to Selene about his wife who was killed in the purge, watch the buildings in the background as he says "she told me she loved me, and then she let the sunlight in". The vehicle used to tow the car that they're sitting in and the camera rig are reflected in the buildings' windows.
Continuity mistake: Last scene of the movie on the roof Selene goes through her prologue looking out at the city and she has a bloody mark over her left eyebrow. Eve reaches for her hand and she looks down; when she looks back up the bloody mark is gone.
Continuity mistake: Selene holds an Antigen scientist in his apartment against a huge window by her left hand and asks about Subject 2. The man tries to break her grip and has his two hands around her wrist. The position of the hands differs between shots showing his face and shots from the side.
Continuity mistake: When Selene escapes the facility at the beginning of the film and slashes some of the guards' throats with a scalpel, she leaves a ton of blood on the floor and walls, as well as a trail of blood as she slides across the floor. But moments later when we see the bodies on a security camera, all of the blood has disappeared.