This film begins two years after the events in The Bourne Identity. Bourne and his girlfriend from the first film, Marie, are living in Goa, India. Bourne is having flashbacks of an assassination in a Berlin hotel. Meanwhile, in Berlin, CIA officers under Deputy Director Pamela Landy are paying $3 million for the "Neski files," documents on the theft of $20 million seven years earlier. An assassin, Kirill, plants Jason Bourne's fingerprints and proceeds to kill the two agents and steal the files and money, giving them to Russian oil magnate Yuri Gretkov. Kirill arrives in Goa to kill Bourne, but Bourne spots him and flees with Marie. As he and Marie drive away, Kirill attempts to shoot Bourne, but instead hits and kills Marie. When the car crashes off a bridge into the river, Kirill believes that Bourne is dead. However, he narrowly escapes and leaves for Naples with money and passports in an attempt to learn why he is again being targeted and to put an end to Treadstone's pursuit of him for good.
In Berlin, Landy finds the planted fingerprints and goes to CIA Headquarters to find out whose they are. She discovers that they belong to Bourne and asks Abbott about Treadstone. He admits that he had Conklin killed after a failed mission and has no idea where Bourne is. Landy tells Abbott that the CIA agent who stole the $20 million was named in the stolen files. A Russian politician, Vladimir Neski, was about to identify the thief when he was murdered by his wife in a Berlin hotel. Landy believes that Bourne and Conklin were involved, and, based on the prints, Bourne was the assassin in Berlin. Both Abbott and Landy go to Berlin to find Bourne and take him down.
In Naples, Bourne uses a known passport, causing him to be identified by security. He incapacitates his interrogator and copies the SIM card on his cell phone. He uses it to listen in on a call from Landy, learning her name, phone number, and that he is suspected in several deaths. He then steals a car and heads to Germany. In Munich, he breaks into another Treadstone agent's house. The agent, Jarda, tells him that Treadstone was shut down after Conklin's murder. He and Bourne are the only ones left. After a struggle, Bourne is forced to kill him and blows up his house to escape from arriving agents.
On their way to Berlin, Landy and Abbott meet Nicky Parsons, Treadstone's handler in Paris. Bourne follows them, believing that Landy ordered the hit in Goa. He targets her with a rifle from a neighboring roof, and calls her to ask why people are after him again. She tells him that it is because he killed two people in Berlin, and he thinks she is talking about the people in his flashbacks. He arranges to meet Nicky at the Alexanderplatz, where he uses a crowded anti-capitalist protest to evade surveillance. He then kidnaps and interrogates her, discovering that Abbott was, in fact, the head of Treadstone, not Conklin. Bourne's flashbacks of Neski's assassination at the Brecker Hotel return, but Nicky knows nothing about it. Upon hearing their conversation over Nicky's transmitter, Landy begins to believe that Bourne is being framed.
Bourne visits the Brecker Hotel and begins to remember more of his mission. He killed Neski on Conklin's orders. When Neski's wife unexpectedly showed up, he shot her to make it look like a murder-suicide. Abbott kills his assistant Danny Zorn upon Zorn's private revelation to him of evidence that Bourne is innocent. Bourne breaks into Abbott's hotel room and records a conversation between him and Gretkov that incriminates Abbott and Gretkov in the theft of the money. Abbott also confesses that he ordered the assassination in Goa, Neski's murder by Bourne, the murder of the agents by Kirill, and the planted fingerprints at the scene. When Landy confronts Abbott, he kills himself, and Bourne sends the tape of the confession to Landy.
Bourne goes to Moscow to find Neski's daughter, where he is shot by Kirill. After an extended high-speed chase through Moscow, Bourne forces Kirill to crash -- killing him. Bourne proceeds to the apartment of Neski's daughter, where he informs her that her mother did not kill her father then commit suicide; Bourne had killed them both on a mission gone wrong. He apologizes and leaves. Gretkov is arrested by Landy, using the evidence she got from Bourne. Some time later, in New York, she receives a call from Bourne, where she expresses her thanks for the tape, and tells Bourne his real name is David Webb and that he was born 4/15/71 in Nixa, Missouri. Bourne finishes the conversation by telling her that she looks tired, indicating that he is looking right at her. The film then ends with Bourne fading into a New York City crowd to the tune of "Extreme Ways" by Moby.