Insectivorous little brown bats are exposed to elevated concentrations of mercury (Hg) through their preference for aquatic-based prey. Here we investigated spatial patterns of total Hg (THg) in fur from 10 little brown bat maternity colonies across Nova Scotia, and assessed relationships with the acidity of nearby lakes and rivers. Total Hg concentrations were measured in fur samples from 149 adult female little brown bats. Values showed significant variation among colonies (mean range 3.76-27.38 μg/g, dry weight), and 48% of individuals had Hg concentrations in excess of the 10 μg/g threshold associated with neurochemical changes in Chiroptera conspecifics (n = 26) from Virginia. Average surface water acidity parameters (pH and acid neutralization capacity) within an 8 km radius of each maternity roost showed strong negative associations with average colony fur THg concentrations. This suggests that freshwater acidity in foraging grounds explains much of the variation in average fur THg concentrations in little brown bat colonies. These findings highlight the significant role that water quality may have on Hg bioaccumulation within terrestrial species that feed on aquatic prey.