The southeastern myotis is nocturnal, resting in hollow trees and Spanish moss during the day. At night, it emerges to forage primarily over water sources like ponds, streams, and wetlands. This species flies slowly and erratically, catching insects in flight or gleaning them from vegetation.
M. austroriparius uses echolocation to navigate and hunt prey. It produces broadband frequency sweeps from 80 to 40 kHz. calls are steep and rapid, at a repetition rate of 18 calls per second. The southeastern myotis roosts in colonies ranging from a dozen to hundreds of individuals.