Seminole bats are aerial insectivorous. They feed in early evening, while in flight, on flies, beetles, true bugs, dragonflies, bees, wasps, and crickets. They are quick and direct when flying, feeding mostly among or above the tops of trees at about 6 to 15 m (Wilson and Ruff 1999). However, it is not uncommon to see them over open ponds, along forest edges, or near lights, presumably where insects accumulate. Like most microchiropterans, seminole bats find their insect prey through echolocation.
Generally, seminole bats are found in lowland forest stands of mixed deciduous and pine trees. Although several bats may be found hanging together, roosting alone is more common. They hang 1.5 to 6.1 m above the ground on the southwest side of trees, clinging to the inside of clumps of Spanish moss (Tillandsia usneoides), leaves, or loose bark; occasionally they can roost in caves. Roosting is more common in pine trees, especially during parturition and lactation. Trees that are selected for roosting are taller and larger in diameter than other trees near the roost area. Selected trees are also tend to be near forest edges that permit easy flight paths (Constantine 1958, 1966; Barbour and Davis 1969; Menzel et al. 1998). The area directly below the moss where they hang is clear of other branches, allowing these bats to drop down to begin their flight. The ground beneath them is covered with leaf litter and other organic debris, thus reducing the amount of sunlight that is reflected on them when hanging.

Lasiurus seminolus is distributed throughout southeastern United States, from east Texas and southeastern Oklahoma eastward to Florida and then northward to eastern North Carolina and Virginia, with extralimital records in Pennsylvania and New York; it is also found in N Veracruz (Mexico) and Bermuda (Simmons 2005).

Several conservation and research actions are needed to assure protection of this species through its distribution. The species is found in a number of protected areas.