In 1560, several scores of Spanish conquistadors, and a hundred enslaved Indigenous people, march down from the newly conquered Inca Empire in the Andes mountains into the jungles to the east, in search of the fabled country of El Dorado. Under the command of Gonzalo Pizarro (Alejandro Repullés), the men, clad in half armor, pull cannons down narrow mountainous paths and through dense, muddy jungle. On New Year's Eve, reaching the end of his supplies and unable to go on without more information, Pizarro orders a group of forty men to scout ahead by raft down river. If they do not return to the main party within one week with news of what lies beyond, they will be considered lost. Pizarro chooses Don Pedro de Ursúa (Ruy Guerra) as the commander of the expedition, Don Lope de Aguirre (Klaus Kinski) as his second-in-command, fat nobleman Don Fernando de Guzmán (Peter Berling) to represent The Royal House of Spain, and Brother Gaspar de Carvajal (Del Negro) to bring the word of God. Also accompanying the expedition, against Pizarro's better judgment, are Ursúa's mistress, Doña Inés (Helena Rojo) and Aguirre's teenage daughter, Flores (Cecilia Rivera, in her only film role).
Traveling through rapids, one of the four rafts gets caught in an eddy, and the others are unable to help free it. That night, gunfire erupts on the trapped raft; in the morning the men on board are found dead, with two missing. Ursúa wants the bodies to be brought back to camp for proper burial. Knowing this would slow down the expedition, Aguirre hints to Perucho (Daniel Ades) to "keep the rust off the cannon". Perucho proceeds to fire the cannon at the raft, destroying it and throwing the bodies into the river.
During the night, the remaining rafts are swept away by the rising river. Time has run out for the scouting mission, and Ursúa decides to return to Pizarro's group. Aguirre leads a mutiny against Ursúa, telling the men that untold riches await them ahead, and reminding them that Hernán Cortés won an empire in Mexico by disobeying orders. Ursúa attempts to put Aguirre in chains, but he and a soldier loyal to him are shot. Inés cares for Ursúa. Aguirre coerces the soldiers to elect the fat, lazy Don de Guzmán as the new leader of the expedition. Aguirre proclaims de Guzmán emperor of the new country and declares Philip II dethroned. A farcical trial of Ursúa results in his being sentenced to death, but de Guzmán surprises Aguirre by granting Ursúa clemency.
Aguirre proves to be an oppressive leader, so terrifying that few protest his leadership. Only Inés has the courage to speak out against him. Knowing that some of the soldiers are still loyal to Ursúa, Aguirre simply ignores her.
The expedition continues on a single, newly built, large raft. An Indigenous couple approaching peacefully by canoe are captured by the explorers, and when the man expresses confusion when presented with a Bible, Brother Carvajal kills them for blasphemy. De Guzmán dines on the low food supplies while the men starve, and has the expedition's only remaining horse pushed off the raft because it annoys him. Soon afterwards he is found garotted near the raft's outhouse. After de Guzmán's death, Aguirre proclaims himself leader. Ursúa is then taken ashore and hanged in the jungle. The group attacks an indigenous village, where several soldiers are killed by spears and arrows. The distraught Inés walks into the jungle and disappears.
On the raft again, the group of slowly starving, feverish men begin disbelieving everything they see, even when shot with arrows. The group stares in disbelief at a wooden ship perched in the highest branches of a tall tree, which Aguirre orders be brought down and refurbished, but Brother Carvajal refuses. In a series of final attacks by unseen assailants, the remaining survivors including Aguirre's daughter are killed by arrows. Aguirre alone remains alive on the slowly drifting raft. The raft becomes overrun by monkeys. The crazed Aguirre tells them: "I, the Wrath of God, will marry my own daughter, and with her I will found the purest dynasty the world has ever seen. Together, we shall rule this entire continent. We shall endure. I am the Wrath of God ... who else is with me?" The final shot is of him waiting for the monkeys to respond.
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