Conservation of bats declining from white-nose syndrome (WNS) impacts requires an understanding of both temporal and landscape-level habitat relationships. Traditionally, much of the research on bat ecology has focused on behavior of summer maternity colonies within species’ distribution cores, including that of the endangered northern long-eared bat (Myotis septentrionalis). To further our knowledge of this species, we evaluated multi-season activity patterns in eastern North Carolina and Virginia, including areas where populations were recently discovered. We used passive acoustic monitoring to assess relative and probable activity of northern long-eared bats from October 2016 to August 2021. Northern long-eared bat relative activity was greatest in areas containing greater proportions of woody wetlands and upland pine-dominated evergreen forests. However, the likelihood of recording northern long-eared bats was associated with smaller proportions of woody wetlands and open water resources. Furthermore, we observed a higher probability of recording northern long-eared bats during non-winter seasons. Probable activity was greatest at temperatures between 10 and 25 C, potentially highlighting an optimal thermoneutral zone for the species regionally. Relative activity of northern long-eared bats on the Coastal Plain of Virginia and North Carolina was primarily driven by cover features, whereas probable activity was driven by a combination of cover features, seasonality, and temperature. Therefore, acoustical surveys for this species may be most effective when targeting woody wetlands adjacent to upland forests, particularly upland pine-dominated evergreen stands, during moderate temperatures of non-winter seasons (1 April–15 November). Moreover, conservation of a diverse mosaic of woody wetlands juxtaposed by upland forests may promote both roosting and overwintering habitat, thereby enhancing overwintering survival, maternity colony establishment, and ultimately, successful reproduction of northern long-eared bats.