Episode 1

In 1476 Florence, Giovanni Auditore monologues about the corruption, betrayal and murder hidden behind the enlightenment of the Renaissance, and about his fight to preserve justice, honor, and his family's safety. From the shadows, he quietly watches his family enjoying themselves at dinner, before departing into the moonlit streets of Florence. Outside, he ambushes a party of mercenaries led by Rodrigo Borgia as they attempt to sneak out of the city. Giovanni kills two of the mercenaries and incapacitates the third, but Borgia escapes in the confusion. Giovanni brings the surviving mercenary to Lorenzo de' Medici, where he is tortured for information, revealing a plot to assassinate the Duke of Milan, Galeazzo Maria Sforza, on St Stephen's Day. Giovanni races to Milan but arrives too late to prevent Sforza's death. After killing the assassins, Giovanni searches one of the bodies and finds several coins stamped with the coat of arms of Venice. Returning home, he monologues that Sforza's death has robbed Medici of a powerful ally, but that he knows where to find those responsible.

Episode 2

Giovanni arrives in Venice to search for whoever ordered Sforza's death. His investigation leads him to the Doge's Palace, Venice, where he sees two men—Silvio and Marco Barbarigo—dispatching a courier to deliver a letter to their master in Rome. Giovanni intercepts and overpowers the courier, who commits suicide to avoid interrogation. Giovanni returns to Florence and presents the letter to Lorenzo and Uberto Alberti, Florence's Gonfaloniere of Justice, but it is encrypted. Uberto gets Father Antonio Maffei da Volterra to decode the letter but secretly orders him not to tell anyone of its contents.

After enjoying some time with his family, Giovanni is summoned by Maffei back to Lorenzo's palace. His son Ezio, confused as to why his father must leave so late in the night, asks to come with him, but Giovanni refuses. At the palace, Uberto claims the letter could not be decoded, meaning the only way to see who it is intended for is to deliver it personally. Giovanni volunteers for the mission and departs for Rome. Back in Venice, Borgia and his allies meet to formulate their next move, and pray "May The Father of Understanding be with us!" while arranging their swords in familiar symbol akin the cross pattée—confirming they are Templars.

Episode 3

Giovanni arrives in Rome and delivers the letter, which is passed through the crowd until it reaches Borgia. Borgia goes to the Holy See and gives the letter to Pope Sixtus IV. The pair discuss the fact that Lorenzo is unwilling to bend to the Pope's authority; when Borgia suggests using force, the Pope agrees to support the Pazzi conspiracy to restore order to Florence. After Borgia leaves, Giovanni follows him to St. Peter's Basilica, only to discover it is an ambush. Borgia reveals that he knows Giovanni's name and invites him to join the Templars and live to see "a new world", but Giovanni refuses. Giovanni manages to fight off Borgia's men, but his left hidden blade is broken and he is struck in the chest with a throwing knife by Borgia, who seizes the opportunity to escape.

Back home, while his wife Maria tends to his wounds, Giovanni confesses to her his fear that Sforza's assassination was merely the start of a conspiracy, and that the next blow will strike Florence. Suddenly, Maffei and several guards arrive at the house, asking for Giovanni. His eldest son Federico lies that his father has already left while Giovanni escapes through a hidden passage. In Rome, Borgia and his fellow Templars agree that the main threat to their plans is Giovanni. Borgia says he has a plan to deal with the Assassin, and that with him gone, nothing will stand in their way. Back in Florence, Giovanni prowls the streets—pausing to muse when he sees Ezio flirting with his girlfriend—while monologuing that dark days are approaching Florence and time is running out. He remarks that no matter what happens, he and his sons are "the Auditore da Firenze, and we are Assassins!" The film concludes with the message "The conclusion ... is in your hands", setting the stage for Assassin's Creed II.