The Knights of the Templar Order are seeking out the Apple of Eden, which legend tells contains the seeds that led to the first sin. The Templars wants to seize the Apple for control over free will in a violent world. The Brotherhood of Assassins were the ones set out to destroy the Templars. Aguilar de Nerha (Michael Fassbender) is tasked with stopping the Templars from taking the Apple. He recites the Assassin's Creed: "We work in the dark to serve the light. We are Assassins."
We jump to the 20th century where a young Callum Lynch (Angus Brown) trying to ride his bike across rooftops. After he fails, he goes home and finds his mother Mary (Essie Davis) has been murdered by Cal's father Joseph (Brian Gleeson), who is dressed as an Assassin. Cal runs away from home as his father is pursued by modern day Templars.
It is now the present day. An adult Callum (now played by Michael Fassbender) is headed to his execution after being convicted of murder. He is hooked up to a chair for a lethal injection. Cal experiences trippy imagery before passing out.
Cal wakes up to find Dr. Sophia Rikkin (Marion Cotillard), who tells him that he is legally dead, but has been brought to a facility. Cal runs out of his room and comes across a window overlooking the city in a massive building. Sophia tells Cal that the building belongs to Abstergo Industries. They need Cal for an important experiment.
Callum is taken to a room with a large claw-like mechanism called the Animus. Sophia explains that the Animus will allow Cal to see the memories of his ancestor, Aguilar. After getting hooked into the Animus, Cal finds himself in the Spanish Inquisition as Aguilar. He and his partner Maria (Ariane Labed) are tasked with rescuing the son of Sultan Muhammad XII (Khalid Abdalla), Prince Ahmed of Granada (Kemaal Deen-Ellis), who has been kidnapped by Templars under the order of Tomas de Torquemada (Javier Gutierrez) in order to obtain the Apple from the Sultan. Aguilar strikes when he kills the Templar group's leader with a Hidden Blade concealed in his sleeve. Aguilar and Maria chase after a chariot where Ahmed is being taken. They run the chariot off the street, causing it to slide over a cliff. Aguilar grabs Ahmed and is left dangling before a Templar leader named Ojeda (Hovik Keuchkerian) approaches him. Sophia then takes Callum out of the Animus, leaving him on a literal cliffhanger.
Cal meets other people in the facility who are descendants of Assassins, including Moussa (Michael K. Williams), whose ancestor Baptiste used voodoo against his enemies.
Cal begins to experience hallucinations of Aguilar fighting, at times appearing as though he is attacking Cal. Sophia explains that these reflections are normal as part of being put in the Animus. She states that the Animus is meant to show people who made them what they are.
Sophia and her father Alan Rikkin (Jeremy Irons) are followers of the Templars. They both claim to be seeking the Apple of Eden in order to rid the world of violence. Rikkin meets with a higher-up, Ellen Kaye (Charlotte Rampling), who wants to shut down Rikkin's project due to the lack of success in locating the Apple.
Cal experiences more hallucinations, seeing Aguilar attacking him. Cal dodges Aguilar's attacks, but he ends up striking several Abstergo officials. He is subdued and taken back to the Animus. Now, Aguilar and Maria are captured and detained in a cell with their mentor Benedicto (Carlos Bardem). The Assassins are set to be publicly executed. Benedicto is burned alive, but Aguilar manages to free himself and Maria. They cause explosions by setting off oil barrels and then run over rooftops as the Templars chase after them. Aguilar performs a Leap of Faith, but it causes Callum in the present to be caught in a malfunction with the Animus.
Cal and Sophia discuss the project as they both hate the Assassins as it was they that killed both their mothers.
Cal reunites with Joseph (now played by Brendan Gleeson). Cal still hates Joseph for killing Mary, but Joseph explains that Mary had willingly allowed herself to be killed so that Abstergo would not experiment on them.
When Callum returns to the Animus, we see that Aguilar and Maria found the Apple about to be exchanged between Torquemada and the Sultan in order for Torquemada to return Ahmed to his father. The Assassins fight the Templars and kill them all except for Torquemada and Ojeda. Aguilar gets the Apple, but Ojeda holds Maria with a knife to her neck. Ojeda demands the Apple, but Maria would rather sacrifice herself than to let Aguilar give up the Apple. Aguilar holds onto the Apple, and Ojeda drives the knife into Maria's neck, killing her. Aguilar fights Ojeda and stabs him to death with his Hidden Blade before making off the with Apple.
The Rikkins learn that the Apple is currently in the tomb of Christopher Columbus. After retrieving it, they head to a temple in London for a grand ceremony. Sophia learns that her father cares more about completely eradicating the Assassins instead of their strive for peace.
Callum has fully synchronized with Aguilar and has accepted his role as an Assassin, especially after seeing Mary dressed as an Assassin as she speaks to him. He leads Moussa and several other modern day Assassins against the Templars, slaughtering nearly all of them. Cal and Sophia come across each other. She threatens to expose him, but he knows she won't do it. They make it to Rikkin and kill him before taking back the Apple.
The Assassins are now free from Abstergo, but Sophia appears to conspire against Callum in retaliation for her father's death.