Abstract Background Emerging infectious diseases in wildlife are an increasing threat to global biodiversity. White-nose syndrome (WNS) in bats is one of the most recently emerged infectious diseases in North America, causing massive declines in eastern bat populations. In the Northeast, winter behavior of bats during the hibernation period, such as flying during the day or in cold weather, has been attributed to WNS. However, winter emergence of bats in the southeastern United States, where winters are warmer, has received little attention. The goals of this study were to determine if winter emergence results from infection by Pseudogymnoascus destructans, the causative pathogen of WNS, and to investigate how pathogen load and prevalence vary by species, site, and over time. Results We collected epidermal swab samples from 871 active bats of 10 species captured outside of hibernacula in Tennessee during winters 2012–2013 and 2013–2014. Deoxyribonucleic acid (DNA) from P. destructans was not detected on 54% of these bats, suggesting that winter emergence occurs regardless of fungal infection. Among infected bats, Perimyotis subflavus (tri-colored bats) had the highest mean fungal load, whereas Myotis lucifugus (little brown bats) had the highest infection prevalence of all individuals captured. Less than 18% (n = 59 of 345 individuals sampled) of all M. grisescens (gray bats) captured had detectible P. destructans DNA on their forearms and muzzle. Hibernacula with large M. grisescens populations had lower fungal loads than sites used by other species; however, mean load per species did not significantly differ between M. grisescens and non-M. grisescens sites. Conclusions We found that pathogen load and prevalence were higher on bats captured during winter 2012–2013 than in the following winter, indicating that fungal loads on bats did not increase the longer a site was presumably contaminated. Repeated low-dose exposure, mild temperatures, and availability of prey during winter in the Southeast may provide a regional refuge for surviving bat populations.