The boreal forest is the largest intact forest in the world, and a refuge for species experiencing range retractions as a consequence of climate and landscape change. Yet, large tracts of the boreal forest are threatened by the cumulative impacts of climate change, natural resource extraction, agriculture, and urbanization, perhaps warranting a shift in focus from biodiversity conservation in intact wilderness to that in anthropologically modified landscapes. We investigated landscape features that influence the distribution of the endangered little brown bat (Myotis lucifugus) in an urbanizing boreal landscape at two spatial scales. We hypothesized that little brown bat activity would be influenced by proximity to available building roosts, because roosts are a potential limiting factor for boreal bats. Secondarily, we predicted that bats would use potential foraging habitat, such as waterbodies, and would avoid young, cluttered forests at the landscape scale. We conducted acoustic surveys of bat activity at 210 sites distributed across the study area in Yukon, Canada, within 1-km grid cells. We tested a priori hypotheses with a set of candidate regression models, accounting for spatial autocorrelation. Our hypothesis about the relative importance of anthropogenic roosts was not supported. Little brown bats were equally active in urban areas (high building density) and rural areas (low building density), perhaps because roosts were adequately available throughout the region. Instead, habitat use was driven by the distribution of potential foraging habitat, particularly waterbodies, which are important sources of aerial insect prey. Little brown bats also avoided young (≤100-yr-old) forest at the landscape scale (including areas regenerating after fire), which may have been poor foraging or roosting habitat, and used areas with a smaller agricultural and industrial footprint. Our results suggest that waterbodies and mature forest are important little brown bat habitats that should be protected from urban encroachment. Proactive conservation of important habitat for species at risk is still possible throughout much of the boreal forest, where human densities are comparatively low and the urban footprint is currently small.