In 49 BC, Bayek, a Medjay charged with protecting the Siwa Oasis, is abducted along with his son Khemu by a group of masked men and taken to an underground vault in the Temple of Amun. They give Bayek a dormant Piece of Eden and demand that he use it to open a secret vault. Khemu helps Bayek escape. While struggling with one of the masked men, Bayek inadvertently kills Khemu. One year later, in 48 BC, Bayek has exiled himself to track down the five masked men to take his revenge. After eliminating two targets, Bayek heads for Alexandria to meet Aya, Bayek's wife. Aya reveals that there is only one target left. Bayek identifies the Royal Scribe Eudoros as the last target, but he is disturbed by Eudoros' last words, which imply there are more masked men. Aya directs Bayek to her friend Apollodorus for more information. Apollodorus introduces him to Cleopatra, who confirms that the masked men are members of the Order of the Ancients, which ousted her from the throne and seeks to control Egypt using Ptolemy as their puppet.
Cleopatra gives Bayek four new targets. He tracks them down and kills them while Aya convinces Pompey the Great to ally with Cleopatra. Bayek receives a letter from Aya explaining there are more members of the Order at large, including members of Ptolemy's royal guard who are likely responsible for Khemu's death. Bayek begins to believe that Cleopatra is using him to kill her rivals. Pompey is killed by Lucius Septimius, forcing Bayek and Aya to sneak Cleopatra into the palace to meet Julius Caesar. Cleopatra impresses Caesar and secures his support. Bayek kills Pothinus, "The Scorpion", but is stopped from killing Septimius by Caesar. Aya watches Ptolemy be eaten by crocodiles when he tries to flee across the Nile.
Cleopatra takes the throne as Pharaoh. Septimius becomes an advisor to Caesar, and Cleopatra cuts ties with Bayek and Aya. Bayek and his supporters realize that Cleopatra and Caesar have now allied themselves with the Order. Bayek gathers his allies to form a brotherhood to counter the Order and defend the people's free will. Bayek and Aya realize the Order showed interest in the tomb of Alexander the Great where they find a mortally wounded Apollodorus. He warns them Caesar's lieutenant Flavius is the true leader of the Order. He and Septimius had taken the Orb and a Staff from the tomb and are going to Siwa to open the Vault.
Bayek tracks Flavius to Cyrene, where he has used the activated Apple of Eden to hold the population in thrall. He kills Flavius, avenging Khemu's death and returns to Aya. Aya recruits Marcus Junius Brutus the Younger and Gaius Cassius Longinus to their cause and heads to Rome to assassinate Caesar and Septimius. Bayek and Aya part ways but form the Hidden Ones, the foundation of the Assassin Brotherhood when they both swear to protect the world from the shadows. In Rome, Aya confronts and kills Septimius, who wields the Staff of Eden. She infiltrates the Roman Senate and assassinates Caesar. Later, she meets Cleopatra and warns her to be a fair ruler or she will return to assassinate her. Afterward, Bayek and Aya (now calling herself Amunet)[a] begin recruiting and training other Hidden Ones as they build the Brotherhood in Egypt and Rome, respectively.
In the present day, Layla Hassan, a former researcher for Abstergo Industries, is tasked with finding and retrieving an artifact in Egypt for the Assassin Brotherhood. Instead, she finds a tomb containing the mummies of Bayek and Aya. Hoping to find any relevant information that would secure her a position in the company's Animus Project, Layla decides to relive both Bayek and Aya's memories using a modified Animus without telling her superiors. When Layla fails to report in, Abstergo sends a team to kill her, but their plan fails. Layla returns to the Animus but is later wakened by William Miles, the Assassin Mentor and the father of the deceased Desmond Miles. She accepts his offer to work with the Assassins but stops short of actually joining them. The two depart for modern-day Alexandria. In experiencing Bayek's memories, Layla may encounter a series of ancient structures built by the First Civilization. Each contains a message that alludes to Layla playing a pivotal role in an upcoming apocalyptic event.