The game takes place in Louisiana near the end of the French and Indian War, where France's defeat caused the city of New Orleans to be taken over by the Spanish government. The citizens were upset over the transition from French to Spanish control and Spain's new occupation within the city. However, in 1765, the French governor Jean-Jacques Blaise d'Abbadie made a negotiation with Templar Rafael Joaquín de Ferrer to stay as governor of New Orleans. This plot was later uncovered by the Assassin Aveline de Grandpré, who infiltrated the governor's mansion and assassinated him.
Following this, de Ferrer made a deal with a man named Baptiste, who was hoping to defect from the Assassins to the Templar Order. Baptiste pretended to act as the recently deceased François Mackandal, as a ruse to attract followers to his cause. He planned to poison New Orleans' nobles and take control of smuggling operations within the Louisiana Bayou. However, his sole purpose was to force Agaté, Assassin and mentor to Aveline, out of hiding in the bayou. Aveline tracked him down, however, killing his followers and assassinating the false Mackandal.
In 1766, the Templar Antonio de Ulloa arrived in New Orleans, to serve as the Spanish governor. However, he left control of the area to French officials and allowed the French flag to remain over the city. Two years afterwards, he made strict trade restrictions and set up a covert slave-trading operation to transport slaves to a Templar worksite in Mexico, creating a rebellion among French officials and the citizens of New Orleans.
Agaté ordered Aveline to assassinate de Ulloa to eradicate Templar presence in New Orleans. Aveline ambushed de Ulloa's carriage and confronted the governor, who told her that slaves had been taken to Chichén Itzá. However, Aveline spared his life and let Ulloa leave the city, in return for a lens used to decode Templar documents and a map leading her to the Templar worksite in Chichén Itzá. Aveline's act of mercy destroyed any trust Agaté had in his student, as she had disobeyed his explicit orders.
Against her mentor's wishes, Aveline disguised herself as a slave bound for Mexico and made her way to the Templar worksite at Chichén Itzá. Whilst there, she came across a defiant slave who mentioned that Jeanne, Aveline's mother, was stationed in Chichén Itzá. Aveline investigated thoroughly and uncovered a page from Jeanne's diary and a map that lead to an artifact located inside a cenote. Upon exploring this cave system, she came across an ancient chamber filled with ruins from the First Civilization, along with a fragment of the artifact she was searching for, known as the Prophecy Disk.
Shortly after Aveline had acquired the artifact, de Ferrer, who was supervising the Templar worksite at Chichén Itzá, broke through into the chamber. However, Aveline overpowered his soldiers and killed him. At the end of the tunnels, she found Jeanne, who realized that her daughter was an Assassin and believed that Agaté had sent Aveline to kill her. She fled, though not before warning Aveline that the Prophecy Disk should never reach Agaté.
After two years of absence, Aveline returned to New Orleans to find that Spanish control was strong. After speaking to fellow Assassin Gérald Blanc, she learned that someone was bribing Spanish soldiers. Upon investigating, Aveline discovered that a man by the name of Vázquez was using Spanish soldiers to seize control of the bayou and take control of the bayou's smuggling operations, which would eventually lead him to discover the location of Agaté. Believing him to be the "Company Man", the head Templar of Louisiana, who de Ferrer mentioned in Chichén Itzá, Aveline immediately hurried to Agaté's hut to warn him of the threat posed by Vázquez's soldiers. Agaté told her to use the power of voodoo to spook the Spanish soldiers into abandoning their mission. Using her blowpipe, Aveline poisoned enough of Vázquez's soldiers so that the rest believed they were cursed and fled the bayou.
Aveline returned to Chichén Itzá and made peace with her mother, who directed her to a canoe inside another cenote. Aveline navigated her way through the cenote and retrieved the final piece of the Prophecy Disk. She offered to bring her mother back to New Orleans, but Jeanne decided to stay behind to take care of the community, which was now rid of Templar influence.
Aveline returned to New Orleans, continuously working to free slaves in the city. Her stepmother, Madeleine de l'Isle, became aware of her work and asked her to help a slave named George escape north. Aveline found a safe passage through the bayou for George to leave through. Whilst escorting him through the swamp, she met with her smuggler allies Élise Lafleur and Roussillon, helping them to deliver supplies to American patriots who were fighting in the American Revolutionary War.
Vázquez attempted to stop Aveline and her allies by sending Spanish soldiers their way, but failed. Aveline defeated Vázquez's soldiers and ensured George and the supplies reached their destination. After returning to the city, Aveline dressed herself in a noblewoman outfit and attended a governor's ball, in hopes that Vázquez would be there. After speaking with several guests, she located Vázquez and asked him to dance. Using her charm, she managed to assassinate him. However, to her surprise, Vázquez revealed that he was not the Company Man.
Aveline next spoke to Gérald, who told her that a Templar working for the Company Man had been found on the New York frontier. Aveline learned that the Templar was Officer Davidson, a Loyalist soldier of the Ethiopian Regiment. Aveline reached the New York frontier in 1777 and met Connor, a half Mohawk, half British Assassin fighting the Templars during the American Revolution.
Aveline and Connor made their way through the frontier to a fort where Officer Davidson was located. Connor distracted the guards and allowed Aveline to slip inside the fort. She confronted Officer Davidson, shocked to learn that he was actually George, the escaped slave she had helped flee north. He attempted to escape, but Aveline managed to shoot his carriage and prevent his departure. From him, Aveline finally discovered the identity of the Company Man: Madeleine de L'Isle, her stepmother. Upon returning to Louisiana, Aveline hurried to her family's mansion and confronted her stepmother. Madeleine revealed to Aveline that she had secretly manipulated her life, preparing her for induction into the Templar Order, as she believed that they shared a common goal.
Afterwards, Aveline traveled to the bayou and met with Agaté, informing him of Madeleine's identity as the Company Man. Agaté was unable to accept his failure and believing that Aveline had sided with the Templars, attacked her. Aveline overpowered him and attempted to convince him to leave Louisiana for his own safety, but Agaté could not live with the humiliation and instead chose to dive to his death.
Aveline decided to infiltrate the Templars and eradicate it from within. Upon returning to New Orleans, she made her way to the Saint Louis Cathedral and gave Madeleine Agaté's necklace to show her allegiance. Following this, she was inducted into the Templar Order by her stepmother. Aveline then gave her the two halves of the Prophecy Disk, which Madeleine placed on an altar and added another piece to hold the two fragments together. However, she could not make sense of the scrambled messages within. Aveline saw this as her moment to strike and reclaim the Prophecy Disk. She eliminated all the Templars inside the cathedral and assassinated Madeleine.
Now alone inside the cathedral, Aveline stepped up to the altar where the Prophecy Disk had been placed. She connected the locket she wore around her neck, which once belonged to her mother, to the artifact, causing it to display a holographic recording detailing a message from the time of the First Civilization. This message recounted the election of Eve as the leader of the rebellion during the Human-First Civilization War.