Low winter temperatures are a major driver of hibernation and migration in temperate North American bats. Hibernation and migration in turn affect bat mortality via white-nose syndrome and collisions with wind turbines. To describe winter bat acoustic activity across a wide temperature gradient and to understand species-specific responses to low temperatures, we recorded nightly acoustic activity of bats at 15 sites across the state of North Carolina, United States, from December through February 2016 – 2018. Bat acoustic activity was recorded at all sites during both winters. Nightly probability of bat acoustic presence regressed positively on ambient temperature. Nightly probability of presence in Lasionycteris noctivagans (silver-haired bat) and Eptesicus fuscus (big brown bat) regressed negatively on wind speed. The mean probability of presence within the same winter condition was highest for L. noctivagans, followed by E. fuscus, Perimyotis subflavus (tricolored bat), and Lasiurus cinereus (hoary bat). Differences in species' mean body weight and roosting preference explained part of the variation of the species-specific probability of presence. Our results can be used to predict bat acoustic presence for these species across the southeastern United States in winter, and better understand the potential threats to bats such as white-nose syndrome and wind turbine interactions.