This species mates in the fall before hibernation, though due to sperm storage, females do not become pregnant until the spring.

[3] It eats a diverse array of insects, foraging with a slow, erratic flight and navigating via echolocation.

Though once considered one of the most common bat species in its range, its populations have declined rapidly since 2006 with the introduction of the fungal disease white-nose syndrome.

The holotype had been collected in the US state of Georgia by American naturalist John Eatton Le Conte.

[9][10][1] Its common name was changed from "eastern pipistrelle" to "tricolored bat" to reflect its revised classification.

Instead, they referred to it as the "perimyotine group", which they gave as the most basal member of a clade that also included the following tribes: Nycticeiini, Eptesicini, Vespertilioni, and another unnamed tribe referred to as the "hysugine group" (including Chalinolobus, Hypsugo, Laephotis, Neoromicia, Nycticeinops, Tylonycteris, and Vespadelus).

Gestation (pregnancy) length is about forty-four days, with females giving birth in June or July.

[15] During the summer, the tricolored bat will roost in tree foliage or buildings, with females alone or in maternity colonies of up to thirty individuals.

Usnic acid, which has anti-insect and anti-bacterial properties, naturally occurs in beard lichens, and no ectoparasites (external parasites) have been documented on the tricolored bat in Nova Scotia.

[21] In another abandoned mine in the US state of Indiana, researchers found that the tricolored bat roosts solitarily during hibernation for the majority of the time (96.8% of observations were singletons).

[17] The tricolored bat's natural predators include the northern leopard frog, birds of prey, raccoons, snakes, skunks, and prairie voles.

[15][24] The tricolored bat is the host to several species of endoparasites (internal parasites) and ectoparasites.

[25] The tricolored bat has experienced severe population decline as a result of the fungal disease white-nose syndrome, which arrived in the US in 2006, with losses of 70% and greater detected in multiple US states.

[26] The disease kills bats by colonizing their skin during the winter, causing them to arouse from torpor and burn through their limited fat reserves.

Though its population experienced dramatic reduction, subsequent studies have found that their numbers may be stabilizing, though hibernacula where many individuals once roosted may only host fewer than five bats, or even one solitarily.

Its range has expanded since the 1980s, with westward expansion reaching the US states of Colorado, New Mexico, South Dakota, and Wyoming.

In the time between the two assessments, the fungal disease white-nose syndrome became widespread in eastern North America, severely impacting the tricolored bat.