With dramatic declines of bat populations due to mortality caused by Pseudogymnoascus destructans (White-nose Syndrome), assessing habitat preferences of bats in the northeastern US is now critical to guide the development of regional conservation efforts. In the summer of 2012, we conducted fixed-station simultaneous telemetry to determine nocturnal spatial use and fixed-kernel home-range estimates of available habitat of a Myotis lucifugus (Le Conte) (Little Brown Bat) maternity colony in an artificial bat house. In summers of 2011 and 2012, we also deployed a 52-ha grid of 4 × 4 Anabat acoustic detectors over five 6–8-day sampling periods in various riparian and non-riparian environments in close proximity to the same bat house. The mean telemetry home range of 143 ha for bats (n = 7) completely overlapped the acoustic grid. Rankings of habitats from telemetry data for these 7 bats and 5 additional bats not included in home-range calculations but added for habitat-use measures (n = 13) revealed a higher proportional use of forested riparian habitats than other types at the landscape scale. Pair-wise comparisons of habitats indicated that bats were found significantly closer to forested riparian habitats and forests than to open water, developed areas, fields, shrublands, or wetland habitats at the landscape scale. Acoustic sampling showed that naïve occupancy was 0.8 and 0.6 and mean nightly detection probabilities were 0.23 and 0.08 at riparian and non-riparian sites, respectively. Our findings suggest that Little Brown Bats select forested riparian and forested habitats for foraging at the landscape scale but may be most easily detected acoustically at riparian sites when a simple occupancy determination for an area is required.