A subarctic fish community in mine-impacted Yellowknife Bay (Great Slave Lake, Northwest Territories, Canada) was investigated for biological and ecological processes controlling arsenic bioaccumulation. Total concentrations of arsenic, antimony, and metals were measured in over 400 fishes representing 13 species, and primary producers and consumers were included to characterize food web transfer. Yellowknife Bay had slightly more arsenic in surface waters (~3 μg/L) relative to the main body of Great Slave Lake (<1 μg/L), resulting in two-fold higher total arsenic concentrations in muscle of burbot (Lota lota), lake whitefish (Coregonus clupeaformis), and northern pike (Esox lucius). Other mining-associated contaminants, specifically antimony, lead, and silver, were typically below analytical detection in those fish species. No evidence was found for enhanced bioaccumulation of arsenic in long-lived, slow-growing subarctic fishes. Food web biodilution of total arsenic occurred between primary producers, aquatic invertebrates, and fish, although trophic position did not explain arsenic concentrations among fishes. Pelagic-feeding species had higher total arsenic concentrations compared to littoral fishes. Arsenic accumulated in subarctic fishes to comparable levels as fishes from lakes around the world with similar water arsenic concentrations. This first comprehensive study for a subarctic freshwater food web identified the importance of water exposure, biodilution, and habitat-specific feeding on arsenic bioaccumulation.