The Yellow Anaconda is a generalist carnivore, preying mainly on animals found in wetland and riparian areas throughout its range. Its diet consists of birds, bird eggs, small mammals, turtles, lizards, other snakes, occasional fish or fish carrion, capybara, and caimans (Reed and Rodda, 2009). The Yellow Anaconda is considered an ambush hunter and constrictor. The digestive system is relatively slow and the Yellow Anaconda may eat only every few days or months, depending on the size of the last prey item. The Yellow Anaconda can survive long periods without prey (Reed and Rodda, 2009).

An adult Yellow Anaconda has few natural predators. Humans are the main predator and it is hunted primarily for its skin, although there are varying numbers imported each year for the pet trade. Predators of juveniles and the occasional adult include Crab-eating Foxes (Cerdocyon thous), tegu lizards (Tupinambis merianae), Spectacled Caimans (Caiman crocodilus), larger anacondas (Eunectes), felids (cats), canids (dogs), procyonids (raccoon family), mustelids (weasel/skunk family), herons, and raptors such as the Crested Caracara (Polyborus plancus) (Reed and Rodda, 2009).

The Yellow Anaconda is a solitary animal, except in breeding season (April and May). Yellow Anacondas have been known to form breeding balls, consisting of one female and multiple males. After a 6-month gestation period, the females give birth to fully developed live young. There is great variation in the literature about litter size, but Reed and Rodda (2009) believed the best estimate for wild individuals was 7–42 with an average of 19.5. These young immediately are able to live on their own. Young anacondas reach sexual maturity at 17 to 29 months old. In captivity, Yellow Anacondas have lived to more than 20 years of age (Reed and Rodda, 2009).