A poacher (Danny Trejo) hides from an anaconda in his boat. While it breaks through the boat, the poacher commits suicide.
While shooting a documentary about a long-lost Indigenous tribe, the Shirishamas, on the Amazon River, director Terri Flores (Jennifer Lopez) and members of her crew—including cameraman Danny Rich (Ice Cube), production manager Denise Kalberg (Kari Wuhrer), her boyfriend, sound engineer Gary Dixon (Owen Wilson), visionary Warren Westridge (Jonathan Hyde), anthropologist Professor Steven Cale (Eric Stoltz), and boat skipper Mateo (Vincent Castellanos)—come across stranded Paraguayan snake hunter Paul Serone (Jon Voight) and help him, believing he knows how to find the tribe they are searching for.
Most of the crew are uncomfortable around Serone, and Cale clashes with him several times in regards to Shirishama lore. Later, while trying to free the boat's propeller from a rope, Cale is stung in the throat by a wasp inside his scuba regulator, leaving him unconscious. Serone performs an emergency cricothyrotomy, seemingly saving Cale's life. 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 giant anaconda he had been tracking........
A team of researchers funded by a New York pharmaceutical firm Wexel Hall, including Dr. Jack Byron, Gordon Mitchell, Sam Rogers, Gail Stern, Cole Burris and Dr. Ben Douglas leave for a jungle in Borneo to search for a "blood orchid", a flower they believe can be used as a type of fountain of youth. Though their guide Captain Bill Johnson and his partner Tran Wu has misgivings about which path to take, Jack convinces him to take an unsafe path. The team goes over a waterfall and has to wade through the river. A giant anaconda emerges from the water and swallows Ben whole, but the rest of the team escape from the river. Bill assures them that it was the largest snake he has ever seen and that it should take weeks for it to grow hungry again. However, most of the team demand that the expedition be called off. They travel to Bill's friend, John Livingston, who lives on the river, to see if Johnson can borrow his boat but they find Livingston dead and his boat crashed.
They find themselves in a small native village consisting of thatched huts and a disemboweled anaconda with a pair of human legs hanging out of the snake's abdomen. The team realizes that the snakes are unusually large in size because their lives have been extended through the orchids, which are a part of the local food chain. Jack says that since they must be close to the orchids, they should press on. However, the others contend that there is no evidence that the orchids will have the same effect on humans. Wanting to leave, they start building an escape raft..............
A baby anaconda, which was captured at the end of the previous film, is being used for experiments by a man named Peter Reysner, who creates a hybrid of the blood orchids from Borneo that enabled the anaconda to grow so large and live so long, and creates a serum for cell regeneration. After the test seemingly works for the baby anaconda to regenerate, Peter burns it. When he disappears, the long anaconda escapes from the cage and kills Peter in a mine filled with blood orchids. J.D. Murdoch, a billionaire suffering from bone cancer, sends his assassin Eugene and his team of henchmen mercenaries to find Peter and the serum Peter created so that it can cure him. He also tells them to be careful of Dr. Amanda Hayes, and to kill her if needed. Dr. Amanda Hayes, the sole survivor of the previous snake attack, and two officers also go in search of Peter, determined to destroy the serum and kill the snake.
On the way, they meet Alex, a trekker who appears lost in the Romanian Carpathians, while doing a pathology project. When they discover the blood orchids in the mine, Amanda sets the explosives to destroy the orchids, but she is attacked by the anaconda that kills two officers, but manages to evade it. As Amanda and Alex leaves the mine, Amanda is knocked unconscious and Alex escapes to get more help. Meanwhile, two groups encounter more people (Scott, Jackson, Patrick, Wendy and Heather) who are looking for the base camp of fellow scientists but get dragged into the search for the snake. Due to the serum, the anaconda can no longer die without significant damage to its internal organs. Heather falls ill due to a spider bite. The next day, most of the people are attacked and eaten by the snake. It first kills one of the hunters working for Murdoch. When Eugene and his henchmen find Hayes and Jackson's group, they capture them. Eugene shoots an already injured Patrick, and also shoots and kills Wendy for trying to escape.
The snake finds them and devours Leila and Hakeem. Amanda and Jackson are forced to find the serum, and are accompanied by two henchmen of Eugene to find it. They put up at Peter's house. There the two find the serum but keep it hidden. The anaconda strikes the house and devours one gunman, and while the other is trying to fight it off, Amanda and Alex escape. Jackie corners them, but is taken by surprise by Jackson. The snake attacks Jackie and he accidentally blows himself up with a grenade while trying to destroy the snake. It then chases Amanda and Scott. Scott sacrifices himself to the snake to buy time for Amanda to run. In an attempt to destroy the snake, Amanda throws a gasoline tank at the snake and blows it up, but the snake regenerates after she flees.............
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