All species of the genus Myotis, including the southeastern bat, rest by day and forage at night.

The feeding flights usually alternate with periods of rest, during which the bats hang to digest their catch.

Disjunct populations of this species occur in the Ohio River Valley of Kentucky, and the majority of the population lives in the northern half of the peninsula of Florida[3] Southeastern myotis are an important food source for barred owls, particularly in the nesting season.

This habitat type has suffered a loss of land cover with the estimation of only 15-25% of pre-colonial forested wetlands remaining in the southeastern United States.

[7] Some habitats are impacted by nearby areas with extensive agricultural development from lack of sufficient buffering.

These inundations can cause drowning of maternity colonies or reduced availability of prey by disrupting larval insects.