Little is known about factors affecting year-round use of roosts by Rafinesque's big-eared bats (Corynorhinus rafinesquii) or the long-term fidelity of this species to anthropogenic or natural roosts. The objectives of this study were to test whether seasonal use of roosts by Rafinesque's big-eared bats varied with roost type and environmental conditions within and among seasons and to document multiannual use of natural and anthropogenic structures by this species. We inspected 4 bridges, 1 building, and 59 tree roosts possessing basal cavity openings; roosts were inspected at least once per week from May through October in every year from 2005 through 2008 and once a month from November through April in every year from 2005 through 2009. We found that use of anthropogenic roosts was significantly greater than the use of tree roosts in summer but that the use of structure types did not differ in other seasons. There was significant seasonal variation in use of anthropogenic and tree roosts. Anthropogenic roost use was higher in summer than in all other seasons. There was no significant difference in tree use among spring, summer, and fall, but use in winter was significantly lower in 2 years of the study. Overall use of anthropogenic and tree roosts was positively related to minimum temperature, but the relationship between use of roosts and minimum temperature varied among seasons. Bats showed multiannual fidelity (≥ 4 years) to all anthropogenic roosts and to some tree roosts, but fidelity of bats to anthropogenic roosts was greater and more consistent than to tree roosts. Our data indicate that Rafinesque's big-eared bats responded differently to environmental conditions among seasons; thus, a variety of structure types and characteristics are necessary for conservation of these bats. We suggest long-term protection of roost structures of all types is necessary for conservation of Rafinesque's big-eared bats in the southeast Coastal Plain.