In 2004, six years after the events of Resident Evil 2 and 3, the Umbrella Corporation’s fiasco in Raccoon City has become a public affair. Following an investigation conducted by the United States Government, several Umbrella officials are implicated and prosecuted. The government formally and indefinitely suspends Umbrella’s business practices, causing the company to become bankrupt.
The game's main protagonist is Leon Scott Kennedy, one of the few remaining survivors of the Raccoon City incident, who was recruited and trained by the US Government to become a special agent after they learned of his actions in Raccoon City. Now an agent for a security agency, Leon is sent on a mission to rescue Ashley Graham, the President’s daughter, who has been kidnapped by a mysterious cult. Leon travels to a Spanish-speaking village in Europe, where he encounters a horde of unruly villagers who pledge their lives to Los Illuminados, the cult that perpetrated Ashley’s kidnapping.
During the course of the mission, Leon is reunited with Ada Wong, a spy Leon met in Resident Evil 2, and Jack Krauser, one of Leon’s former colleagues, thought to be dead. He also meets Luis Sera, a former Los Illuminados’ researcher, who aids Leon on his mission before he is killed. By examining Sera’s notes, Leon discovers that Los Illuminados gained control of their subjects by implanting a mind controlling parasite known as Las Plagas into their bodies.
After Osmund Saddler, the leader of the cult, discovers that Leon has rescued Ashley, he commands his subjects to use any means to recover her. A surprise attack by Saddler’s airborne minions leads to Ashley’s recapture, forcing Leon to travel to a military and research complex located on a nearby island. After numerous altercations with Saddler's forces, Leon is able to successfully rescue Ashley, and defeat Saddler with Ada's assistance.
Leon recovers a vial containing a Plagas sample from Saddler’s corpse, but Ada forces him to give it to her. She then escapes from the complex in a helicopter, leaving Leon and Ashley to escape via jet-ski. Although Leon and Ashley manage to escape from the island, the sample's whereabouts and fate remain uncertain.