Predation data for this species is limited due to its cryptic nature. Giant river otters are known to prey on adult matamatas in Guyana, sometimes bringing them ashore and killing them. Eggs are likely vulnerable to tegu lizards (Tupinambis teguixin), which have been identified as the main natural predator of matamata eggs in Venezuela.

Fish like piranhas may cause minor injuries—in one Venezuelan study, most adult matamatas captured showed healed mutilations (missing toes, occasionally feet or cephalic skin flaps), potentially inflicted by the abundant piranha sharing their habitat. Hatchlings and juveniles are presumably more vulnerable to a wider range of aquatic and terrestrial predators, though specific predation records are lacking.

Mata matas don't hibernate in the traditional sense. These tropical turtles remain active year-round in their warm, equatorial habitat. However, their activity levels likely fluctuate with seasonal changes in water levels. In the Venezuelan llanos, where dramatic wet-dry seasonal cycles occur, turtles may become concentrated in shrinking pools during the dry season, making them temporarily more visible to observers. During high water periods, they disperse into flooded forests and become much harder to find.

Mata matas are specialized ambush predators that feed almost exclusively on live fish. They sit motionless on murky river bottoms, their camouflaged bodies blending with dead leaves, waiting for prey to swim within range. The elaborate skin flaps on their head are highly sensitive to water movements, allowing them to detect approaching fish even in total darkness.

When prey comes close enough, the turtle strikes with explosive speed—thrusting its head sideways while simultaneously opening its cavernous mouth and expanding its throat. This creates a powerful suction that vacuums the fish into its mouth along with a rush of water, which is then expelled as the prey is swallowed whole. In French Guiana, the characin fish Erythrinus erythrinus (typically 20–25 cm long) is commonly taken prey. While fish dominate the diet, they may occasionally take aquatic invertebrates, and some sources suggest small birds or mammals that enter the water could be taken opportunistically.

Nesting occurs in the early dry season, typically October through December, when water levels begin to drop. Females seek out sandy or clayey banks rather than open beaches, often climbing steep riverbanks in a sideways fashion to reach suitable nest sites.

The nest is dug to about 10 cm depth using the hind legs, and females deposit 12–28 nearly spherical, hard-shelled eggs measuring 34–40 mm in diameter. The eggs require an exceptionally long incubation period—often around 200 days in captivity, though wild incubation may be faster, possibly triggered by contact with rising floodwaters. Hatchlings emerge at roughly 49 mm shell length and weigh 15–19 g.

Sexual maturity is reached at approximately 5–7 years. Males display a more concave plastron than females and have longer tails with more distally positioned vents. Courtship behavior in captivity involves the male approaching the female, making head movements, and synchronized swimming before mating.

Everything about the mata mata is extreme. That bizarre, flattened head with its dangling skin flaps isn't just for show—each flap is packed with nerve endings that detect the slightest water movements, essentially creating a highly sensitive motion-detection array. The tiny eyes are almost irrelevant; this turtle hunts by feel in waters too murky to see through.

The feeding strike is one of nature's most dramatic. The turtle can expand its throat so rapidly that it creates a vacuum, sucking in fish along with a rush of water. The massive hyoid apparatus (throat bones) and heavily muscled neck power this suction feeding—a technique so effective that captive matamatas have been observed herding fish into corners before striking.

Perhaps most remarkable is the species' divergence from its Amazon relative: the two lineages separated approximately 13 million years ago during the late Miocene, around the time the Orinoco Basin was forming as a distinct drainage system. Despite living so close geographically, they've been on separate evolutionary paths longer than humans and chimpanzees.