Environmental contamination and the introduction of non-native species are two anthropogenic disturbances that have profound implications for ecosystems. Mercury (Hg) transfers through food webs where it can bioaccumulate and biomagnify to toxic concentrations. Non-native introductions alter food webs and influence energy and Hg movement. This study examined Hg concentrations and stable isotope compositions in native and non-native fishes among four river subbasins of the upper Colorado River Basin (UCRB) to inform the combined influence of Hg exposure and non-native competition on the conservation status of native fishes. We assessed Hg biomagnification at the fish assemblage level, compared concentrations to risk thresholds for fish health, and evaluated niche overlap. Generalist-invertivores had the lowest Hg concentrations (0.001-0.191 ug g-1 wet weight) and no individuals exceeding the U.S. Environmental Protection Agency aquatic life criterion (0.225 ug g-1). Piscivores had the highest Hg concentrations (0.008-1.840) and 74% of individuals exceeded the criterion. Hg biomagnification was observed across the basin. However, Hg concentrations and trophic magnification varied across subbasins, with higher magnification factors in the Lower Green and White-Yampa subbasins. These subbasins also had the highest proportion of non-natives, suggesting subbasin-specific patterns in Hg cycling across the UCRB. Stable isotopes revealed niche overlap among native and non-native species and trophic position varied spatially within species. Non-native species have likely reshaped food webs and intensified the threat Hg poses to native fish conservation. Continued monitoring is needed to track contamination trends, particularly studies that focus on Hg sources entering the UCRB food web, vectors for accumulation, and competitive interactions among native and non-native species.