The Clinch River watershed of the upper Tennessee River Basin of Virginia and Tennessee, USA supports one of North America's greatest concentrations of freshwater biodiversity, including 46 extant species of native freshwater mussels (Order Unionida), 20 of which are protected as federally endangered. Despite the global biological significance of the Clinch River, mussel populations are declining in some reaches, both in species richness and abundance. The aim of this study was to evaluate the exposure of adult resident mussels to a suite of inorganic and organic contaminant stressors in distinct sections of the Clinch River that encompassed a range of mussel abundance and health. To provide insight into the potential role of pollutants in the decline of mussels, including within a previously documented “zone of mussel decline”, the mainstem Clinch River (8 sites) and its tributaries (4 sites) were examined over two consecutive years. We quantified and related metals and organic contaminant concentrations in mussels to their associated habitat compartments (bed sediment, suspended particulate sediment, pore water, and surface water). We found that concentrations of organic contaminants in resident mussels, particularly the suite of 42 polycyclic aromatic hydrocarbons (PAHs) analyzed, were related to PAH concentrations in all four habitat (media) compartments. Further, PAH concentrations in mussel tissue (range 37.8–978.1 ng/g dry weight in 2012 and 194.3–1073.7 ng/g dry weight in 2013) were negatively related to the spatial pattern in mussel densities (r s = −0.64, p ≤ 0.05 in 2012 and r s = −0.83, p ≤ 0.05 in 2013) within the river, and were highest in the “zone of mussel decline”. In contrast, the suite of 22 metals analyzed in resident mussels were largely unrelated to the spatial pattern of variation of metals in the four habitat compartments except for Manganese (Mn; range 3630.5-23,749.2 μg/g dry weight in 2012 and 1540.4-12,605.8 μg/g dry weight in 2013) in surface water (r s = 0.58, p < 0.1) and pore water (r s = 0.76, p ≤ 0.05). This study revealed that PAHs and Mn are important pollutant stressors to mussels in the Clinch River and that they are largely being delivered through the Guest River tributary watershed. Accordingly, future conservation and management efforts would benefit by identifying, and ideally mitigating, the sources of PAHs, Mn, and other current or legacy mining-associated pollutants to the mainstem river and its tributaries. • Examined the role of pollutants in unionid mussel declines in Clinch River, USA. • Organic and metal contaminants were prevalent in documented zone of mussel decline. • Mining-associated PAHs and manganese are important pollutant stressors to mussels. • Guest River tributary was identified as important source of pollutants to Clinch River. • Mussel populations are likely to decline further, if legacy and ongoing pollution is not addressed.