Most vespertilionids are cave dwellers, but they also shelter in mine shafts, tunnels, old wells, rock crevices, buildings, tree hollows, the foliage of trees and bushes, hollow joints of bamboo, large tropical flowers, tall grass, abandoned bird nests, storm sewers, and culverts, as well as under rocks and loose bark on tree trunks. The members of this family generally pitch head upward when alighting, hanging by their thumbs and toes, then quickly shuffle around until they are hanging head downward by their toes, their wings folded alongside the body. Often they hang on a vertical surface rather than suspend themselves freely. Some members of this family are solitary, others roost in pairs or in small groups, and still others generally shelter in colonies. The colonial forms usually return to the same roosting site year after year.
Some species remain in colonies throughout the year, whereas others congregate only in winter.
A number of forms migrate between summer and winter quarters. Homing ability has been demonstrated in several forms. Vespertilionids send out ultrasonic sounds through their mouths. Nearly all members of this family feed mainly on insects. Myotis vivesi is definitely known to feed on fish, and two other species of Myotis are suspected to do so.