Little brown bats use a wide range of habitats and often use human-made structures for resting and maternity sites. They typically roost in caves and mines in the winter, and they can be found in trees, artificial structures, bat houses, under rocks and in piles of wood in the summer. Foraging habitat requirements are generalized, occurring primarily over streams and other bodies of water, along the margins of lakes and streams or in woodlands near water. Winter hibernation sites like caves, tunnels and abandoned mines generally have a relatively stable temperature of about 2 to 12 Celsius, as documented by T.H. Kunz and J.D. Reichard in 2010. Maternity colonies commonly are in warm sites in buildings, like attics, bat houses, other human structures, and infrequently, in hollow trees. 

The diet of the little brown bat consists of a wide variety of flying insects, including mosquitoes, midges, caddisflies, moths, various hoppers, small beetles and spiders, as documented by J.O. Whitaker, Jr. and B. Lawhead in 1992. The wide and diverse prey selection for this species may have allowed the broad geographic distribution, as noted by L.A. Kaupas and R.M.R. Barclay in 2017. Individuals in the extreme northwest region of the range may consume more terrestrial insects, because flying insects are less available, as documented by R.P. Shively and others in 2018. 

Little brown bats primarily hibernate in caves and cave-like structures. They emerge from hibernation and disperse on the landscape for the summer and early fall. Females begin to form maternity colonies that can consist of two to more than 100 individuals. Females may form maternity colonies in large tree cavities, attics or bat houses. Males often roost under exfoliating bark, within tree trunks and within tree cavities of live and dead trees during the summer. Males have also been observed roosting in human-made structures during the summer.

Large numbers of individuals may swarm around caves or mines in late summer and mating typically occurs in the fall. Females store sperm during the winter, a process known as delayed fertilization. Females typically give birth to a single pup in May or June after 50 to 60 days of gestation. The young are able to fly in about four weeks.

Bats of this species are known to live up to 30 years.