Mercury concentrations remain elevated in sediments and biota of the Wabigoon River downstream from Dryden, Ontario, the home of a former chlor-alkali plant. Understanding the current extent and severity of mercury contamination downstream of this industrial legacy site is of great importance in managing the mercury contamination within the traditional territory of Asubpeeschoseewagong Anishinabek (Grassy Narrows First Nation), located downstream of Dryden. The objective of this study was to use mercury stable isotope ratio analysis to distinguish between legacy mercury from the former chlor-alkali plant and mercury from geogenic sources. Mercury concentrations in surface sediments and biota downstream of the historical source of mercury discharge are elevated relative to the chosen reference location, Wabigoon Lake (WL). Mean sediment mercury levels were as high as 3.27 μg/g at the hydroelectric dam location compared to 0.05–0.10 μg/g at Wabigoon Lake sediments. Isotope ratios in aquatic biota and sediments collected from within the system were distinct from Wabigoon Lake, indicating that anthropogenic mercury contamination is distinguishable from geogenic mercury. Average δ202Hg values of −2.46 ± 0.41 observed in sediments of WL were consistently more negative compared to downstream values, which varied from −1.34 to 0.30 ‰. Young-of-the-year Yellow Perch and Hexagenia were found to have significantly more positive δ202Hg values downstream from Wabigoon Lake.