Day-roost selection by Lasiurine tree bats during winter and their response to dormant season fires is unknown in the southeastern United States where dormant season burning is widely applied. Although fires historically were predominantly growing season, they now occur in the dormant season in this part of the Coastal Plain to support a myriad of stewardship activities, including habitat management for game species. To examine the response of bats to landscape condition and the application of prescribed fire, in the winter of 2019, we mist-netted and affixed radio-transmitters to 16 Lasiurine bats, primarily Seminole bats (Lasiurus seminolus) at Camp Blanding Joint Training Center in northern Florida. We then located day-roost sites to describe roost attributes. For five Seminole bats, one eastern red bat (Lasiurus borealis), and one hoary bat (Lasiurus cinereus), we applied prescribed burns in the roost area to observe bat response in real-time. Generally, Seminole bats selected day-roosts in mesic forest stands with high mean fire return intervals. At the roost tree scale, Seminole day-roosts tended to be larger, taller and in higher canopy dominance classes than surrounding trees. Seminole bats roosted in longleaf (Pinus palustris), slash (Pinus elliotii) and loblolly pine (Pinus taeda) more than expected based on availability, whereas sweetbay (Magnolia virginiana), water oak (Quercus nigra) and turkey oak (Quercus laevis), were roosted in less than expected based on availability. Of the seven roosts subjected to prescribed burns, only one male Seminole bat and one male eastern red bat evacuated during or immediately following burning. In both cases, these bats had day-roosted at heights lower than the majority of other day-roosts observed during our study. Our results suggest Seminole bats choose winter day-roosts that both maximize solar exposure and minimize risks associated with fire. Nonetheless, because selected day-roosts largely were fire-dependent or tolerant tree species, application of fire does need to periodically occur to promote recruitment and retention of suitable roost sites.