Freshwater mussels are among the most sensitive species to a variety of chemicals in water exposures. However, few studies have been conducted to evaluate the effect of toxicants in sediments on mussels. Industrial discharges containing polyaromatic hydrocarbons (PAHs), volatile organic compounds (VOCs), and metals entered the Kanawha River surrounding Blaine Island, South Charleston, West Virginia, USA; a river which supports eight federally endangered mussel species. We collected sediment samples from a highly contaminated site, a nearby upstream site, and a further upstream reference site to assess the effects of contaminated sediment on the survival and growth of a unionid mussel (fatmucket, Lampsilis siliquoidea) and a commonly tested benthic organism (amphipod, Hyalella azteca) using standard 28-d sediment toxicity tests. We also determined mussel toxicity in a serial dilution of the highly contaminated sediment. Results showed that concentrations of PAHs, VOCs, and metals in the contaminated sediment were consistently greater than the other two sites. The mean survival of mussels and amphipods in the reference sediment was 100% and 95%, respectively, whereas the mean survival of both test species in the contaminated sediment was 0%. In the sediment dilution study, mean survival and biomass of mussels in the ≥6.25% treatment were significantly reduced relative to the control, with a 25% inhibition concentration of 4.1% for survival and 3.6% for biomass. We used sediment screening values and equilibrium partitioning sediment benchmarks to determine that nickel, mercury, and PAH mixture were likely responsible for the toxicity observed to mussels and amphipods and will provide critical data to identify and mitigate the sources of the mixture in contaminated sediment.