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In the previous film, former security operative Alice and environmental activist Matt Addison fought to escape an underground genetic research facility called the Hive, the source of a zombie outbreak. The pair were part of an attempt to expose illegal experiments being performed there by the pharmaceutical company Umbrella Corporation. The film ended as Alice and Addison were taken into custody by Umbrella and separated.

Umbrella sends a team to the Hive to investigate what happened there; the team is overrun by zombies who quickly spread to the nearby Raccoon City. Umbrella reacts by quarantining the city and evacuating vital personnel from it. Angela Ashford, the daughter of Umbrella researcher Dr. Charles Ashford, goes missing after her security car is involved in a collision while being extracted from the city. Meanwhile, disgraced Raccoon City Police Department Special Tactics And Rescue Squad (STARS) operative Jill Valentine returns to her former precinct to encourage her fellow officers to evacuate Raccoon City. Alice awakens in a deserted hospital and wanders the city in search of supplies, while Umbrella uses the only bridge out of the area to evacuate civilians. At the bridge, Valentine meets with her former partner, Sgt. Payton Wells. A civilian turns into a zombie, biting and infecting Wells. In response to the virus reaching the bridge, Major Timothy Cain, leader of the Umbrella forces in Raccoon City, seals the exit and forces the residents to return to the city.