AbstractMany cave-roosting bats are declining in number throughout their range because of multiple threats, including disease, disturbance by humans, and habitat loss. Successful conservation of cave-roosting bats requires an understanding of the roles of microclimate, cave structure, and the surrounding landscape on the distribution and abundance of bats, and in particular, the use of winter cave roosts. Cave bats have been well studied in temperate climates, but we know little about these bats in subtropical climates. From 2015 to 2017, we conducted 399 winter surveys of 162 caves in Florida to evaluate factors affecting the presence and abundance of bats at cave roosts. We also determined whether temperatures at these cave roosts were suitable for Pseudogymnoascus destructans, the fungus that causes white-nose syndrome. Across all 3 y, we detected three species of bats: tricolored bat Perimyotis subflavus in 126 (77.8%) caves, southeastern myotis Myotis austroriparius in 51 (31.5%) caves, and a single Rafinesque's big-eared bat Corynorhinus rafinesquii. We analyzed the presence of both tricolored bats and southeastern myotis in caves by using mixed-effects models with a binomial distribution and the number of tricolored bats by using generalized linear mixed-effects models with a Poisson error distribution. Tricolored bat presence was significantly and positively influenced by a cooler cave surface temperature, larger cave entrance size, a single entrance, and the presence of solution holes. Tricolored bat abundance increased in longer, cooler caves that had solution holes, a single, unobstructed entrance, and had no signs of flooding. Southeastern myotis presence was positively associated with longer, domed caves. To be most effective, conservation efforts in subtropical climates should focus on caves with the identified characteristics. More than 90% of caves surveyed each year had a cave interior surface temperature below the upper critical growth limit for P. destructans (19.8°C), indicating that most Florida caves are suitable for growth of P. destructans. Therefore, regular monitoring of winter bat populations in subtropical caves may be important for detecting P. destructans and white-nose syndrome.