Suitable hibernacula for bats are currently rare in the Appalachian region of the USA. While hibernacula are often degraded by anthropogenic activities, some actions have created winter habitat, fostering opportunities for partnerships. For example, abandoned railroad tunnels often provide winter habitat, and are sometimes publicly owned. Our goal was to locate abandoned railroad tunnels throughout Ohio, the West Virginia panhandle, and western Pennsylvania, USA, evaluate their suitability for bats, and improve habitat where possible. We surveyed 75 tunnels from 2017–2022 and collected data on microclimates, physical dimensions, and use by bats. Six sites (8%) were used by Myotis lucifugus, 16 (21% by Perimyotis subflavus, and 39 (52%) by Eptesicus fuscus. Although M. lucifugus were rarely found, tunnels were the only hibernacula known to be used by the species in Ohio. Furthermore, the largest winter colonies of P. subflavus in Ohio were found in tunnels. Tunnels occupied by both species were partially sealed and flooded, with internal temperatures that varied by < 5 °C during winter. By contrast, E. fuscus commonly inhabited tunnels that were unsealed and experienced large temperature fluctuations. Finally, two tunnels were gated by the Ohio Department of Transportation as the start of a conservation program. One site had populations of M. lucifugus and P. subflavus that were declining and rebounded following gating. The second was also modified to stabilize internal temperatures. These results highlight the value of abandoned railroad tunnels for bats and provide an example of collaborative stewardship headed by a government transportation authority.