Environmental contaminants and non-native species introductions negatively affect aquatic ecosystem conservation. Mercury (Hg) accumulates within food webs where it can biomagnify to toxic concentrations, which can be affected by altered trophic relationships from non-native species. This study examined Hg concentrations (n samples = 655) and stable carbon and nitrogen isotope (n = 746) compositions in native (n = 313) and non-native (n = 342) species among four river subbasins (Gunnison, Lower Green, Colorado-Dolores, and White-Yampa), in the Upper Colorado River Basin (UCRB) during 2016 and 2017 to inform the potential combined effects of Hg exposure and non-native species interactions on native fishes of conservation concern. We assessed Hg biomagnification at the fish assemblage level, compared concentrations to risk thresholds for fish and human health, and evaluated niche overlap among trophic levels across subbasins. The generalist-invertivores feeding guild had the lowest Hg concentrations (0.001–0.191 μg g⁻¹ wet weight), and no individuals exceeded the U.S. Environmental Protection Agency aquatic life criterion (0.225 μg g⁻¹). Piscivores had the highest concentrations (0.008–1.840 μg g⁻¹), and 74% of individuals exceeded the criterion. Biomagnification across trophic levels was observed throughout the UCRB, yet varied by subbasin, with higher magnification factors in the Lower Green and White-Yampa subbasins which also had the highest proportion of non-native species. Stable isotope compositions revealed niche overlap among native and non-native species. Additionally, trophic position varied within species among the subbasins. The proliferation of non-native species has likely reshaped food webs and may have intensified the potential threat Hg poses to native fish recovery and conservation. This study documented increased Hg concentrations relative to past assessments, suggesting that continued monitoring could assist in evaluating trends in Hg accumulation. Particularly studies that focus on sources entering the food web, vectors for accumulation, and competitive interactions among native and non-native species.