Roads have variable impacts on wildlife, including bats. Bat habitat preferences along roads vary across spatial scales, creating the need for multi-scale habitat studies. The main objective of this study was to determine what habitat factors impact bat activity and species richness along road transects at local and landscape scales and to identify areas of highest bat activity in the Oak Openings Region of Northwest Ohio using acoustic monitoring. At the local scale, bat activity and/or species richness were positively associated with canopy height, clutter at 0-3 meters, humidity, natural habitat, canopy cover, months water present, and temperature, and were negatively associated with clutter at 3-6.5 meters. At the landscape scale, activity and/or richness had positive associations with habitat heterogeneity, roads, open natural habitats, ponds, upland prairie, and overall forest cover, and had negative associations with conifers and cropland. High activity areas were consistent between years and clustered in the northern part of the region, especially near protected areas and low activity areas were typically located in the southern part of the region near agricultural areas. When managing bat habitat along roads, it would be advantageous to decrease mid-level clutter, vary habitat types, increase savanna and upland prairie cover, avoid excessive conifer or cropland cover, plant tall trees, and provide water sources. This study shows that combining acoustic road surveys with habitat analyses at multiple scales can be used to examine the most important habitat factors in uencing bat activity and species richness.