Bat populations may be vulnerable to the barrier impacts of roads, including habitat restriction and traffic mortality. Under-road passageways may reduce these impacts, but little is known about the ecological factors influencing their use by bats. The study area provided a natural experimental design, in that adjacent under-motorway passageways had contrasting dimensions (two long, narrow drainage pipes within <1 km of a large underpass for a minor road), and local bat species had contrasting functional and morphological adaptations. We predicted that inter-species differences in flight capability and sensory perception would influence bat use of passageways. All-night acoustic recordings of bat activity inside passageways (52 nights) indicated clear guild-specific responses to passageway dimensions. Only Rhinolophus hipposideros, Myotis nattereri and Plecotus auritus flew through the narrow drainage pipes. These species are adapted for flight and foraging in cluttered airspace, in terms of wing morphology and echolocation signal design. Edge-space species (Pipistrellus pipistrellus, Pipistrellus pygmaeus) were highly active in the area but never flew through the narrow pipes. All species, except the open-adapted Nyctalus leisleri, flew through the large underpass. Simultaneous recordings made above and below this underpass (16 nights) also indicated that species' tendency to cross over, rather than under, the structure was inversely related to the degree of clutter-adaptation. If motorways are built through bat habitat, trade-offs between optimal mitigation of impacts on protected bats and cost/engineering practicality are inevitable. Large underpasses are advisable where possible as they accommodate a wider range of species, and bats are less likely to fly over them. However, their construction is costly and is dependent on landscape topology. If the target species for mitigation are clutter-adapted bats, our findings indicate that incorporation of a greater number of suitably located small tunnels into new roads may facilitate safe passage more effectively than fewer large underpasses.