Nyctalus noctula is one of the most common bats in the western Palearctic, whereas N. leisleri is relatively rare (except in Ireland, where N. noctula does not occur) and more limited to ancient forests. We radiotracked 26 N. noctula and 25 N. leisleri to 52 and 50 roost trees, respectively, from May to August in 1998–2002 in the Białowieża Primeval Forest in eastern Poland to test the hypothesis that N. leiseri has more specific tree-roosting requirements than N. noctula. Both species selected roosts at the microscale (cavity level), mesoscale (tree level), and megascale (plot level). N. noctula significantly preferred oaks, and avoided hornbeams and alders. N. leisleri roosted more often in oaks and ashes, and avoided hornbeams and alders. Roost trees occupied by both species were thicker and taller, with higher crowns than available trees. N. noctula and N. leisleri used oaks more frequently than ashes when average ambient temperatures were lower. Pregnant and lactating females of both species most often used oaks, whereas after the young could fly most roosts were in other tree species, mainly ashes. Reproductive status influenced the choice of roosts used by N. noctula, whereas both the reproductive status and ambient temperature were influential for N. leisleri. Both taxa preferred dying trees and avoided healthy ones, although N. noctula used hollows in healthy trees significantly more often than N. leisleri. In our opinion, more frequent use of healthy trees by N. noctula suggests a better ability to exploit younger, managed forests. However, differences in roost selection between these species were small.