Tri-colored bats roost in clusters up to hundreds of individuals in tree cavities, Spanish moss, and under loose bark. They emerge at dusk to forage around forest edges and open wetlands. Tri-colored bats have slow, maneuverable flight less than 30 feet above ground as they pursue small insects like moths, mosquitoes, beetles and flies.
Sounds are also important for orientation and social communication. Colonies produce a constant low-intensity vocalization audible to humans. Their echolocation frequencies range from 25 to 50 kHz, often modulated. Tri-colored bats normally roost apart from other bat species.