The story begins on the Amazon River where we see a poacher on a boat desperately trying to get away from something. He climbs the boat's mast with pistol in hand. An attack is looming, his fate unknown.
An exploration crew is shooting a documentary about a long-lost Indian tribe, the Shirishamas, on the Amazon River. The crew come across stranded Paraguayan snake hunter and help him, believing he knows how to find the tribe they are searching for.
The crew includes director Terri Flores (Jennifer Lopez), cameraman Danny Rich (Ice Cube), production manager Denise Kalberg (Kari Wuhrer), sound engineer Gary Dixon (Owen Wilson), visionary Warren Westridge (Jonathan Hyde), anthropologist Professor Steven Cale (Eric Stoltz), and captain of the boat Mateo (Vincent Castellanos). Along with the rescued snake hunter, Paul Serone (Jon Voight).
While trying to free the boat's propeller from a rope, Cale is stung in the throat by a wasp inside his scuba gear. Now unconscious, Terri tends to the gravely ill Cale. With that, Serone takes command of the boat and the crew.
They are then forced to help him achieve his true objective: hunting down and capturing a record-breaking green anaconda he had been tracking. Mateo is the first of the crew to be killed by the anaconda. The anaconda wraps around him and then breaks his neck near a boat where a poacher had been killed at the beginning of the film.
The others try to find him while Gary sides with Serone, who promises if they help him find the anaconda, he will help them get out alive. Gary himself is eaten next when they attempt to capture the anaconda.
The survivors overcome Serone and tie him up. When Denise attempts to kill Serone, he gets the edge and strangles her to death with his legs before dumping her body in the river, later claimed by the river monster. The anaconda returns and kills Westridge.
The snake then coils itself around Danny, only to be shot dead by Terri and Serone incapacitated by the newly awakened Cale, who soon loses consciousness again. Danny punches Serone, knocking him into the river. However, Terri and Danny are soon re-captured when Serone catches up to them.
He dumps a bucket of monkey blood on them and uses them as bait in an attempt to capture a second, larger anaconda. The anaconda wraps around Terri and Danny and begins to suffocate them. They are caught in a net by Serone, but the snake breaks free.
Serone tries to escape, but the anaconda finally manages to coil around him and suffocate him. Terri and Danny cut their bonds and watch in horror as the anaconda slowly swallows Serone whole.
Terri finds a nest of baby anacondas in a building, but the snake arrives. The snake regurgitates the still twitching Serone, then chases Terri up a smoke stack.
The snake becomes trapped in the smoke stack, whereupon Danny ignites a fire below and burns the snake alive. As Terri and Danny recuperate on a nearby dock, the snake appears one final time. Danny beats the anaconda with an axe until it is finally dead.
Afterwards, Terri and Danny reunite with Cale, who begins to revive on the boat. As they float down the river, they accidentally locate the natives for whom they were originally searching. They realize Serone was right and begin filming their documentary as the film ends.