 This species is occasionally observed roosting with Rafinesque's big-eared bats.The range of this species includes southern Illinois and Indiana in the north, westward into southeastern Oklahoma, western Tennessee and Arkansas, and northeastern Texas, and eastward to the southern part of North Carolina.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 

Southeastern myotis are an important food source for barred owls, particularly in the nesting season.