Persistent and bioaccumulative contaminants often reach concentrations that threaten aquatic life by causing alterations in organism behavior and development, disruption of biological processes, reproductive abnormalities, and mortality. The objectives of this research were to determine the aquatic food web structure and trophic transfer and accumulation of contaminants within a riverine ecosystem and identify potential stressors to the health of an imperiled fish, the robust redhorse (Moxostoma robustum) and other species of conservation concern in a large Atlantic Slope (USA) river. Trophic position was determined for food web taxa by stable isotope analyses of representative producers, consumers, and organic matter of the Yadkin-Pee Dee River of North Carolina and South Carolina. Contaminant analyses were performed on water, sediment, organic matter, and aquatic biota to assess the prevalence and accumulation of polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), current use pesticides (CUPs), polycyclic aromatic hydrocarbons (PAHs), and selected metals. Contaminants were prevalent in the environment and food web components of the river. PCBs were detected in 32% of biotic samples (mean 0.24 μg/g dry weight [DW], range 0.01–3.33 μg/g DW), and DDTs (legacy OCPs and metabolites) were detected in 90% (mean 0.014 μg/g DW, range 0.0004–0.29 μg/g DW). The trace metals manganese and cadmium exceeded published threshold effect concentrations in sediment (460 and 0.99 μg/g DW, respectively). Mercury was detected in all food web samples exhibiting a mean of 0.61 μg/g DW and range 0.006–2.35 μg/g DW (mean 0.13 μg/g wet weight [WW], range 0.001–0.6 μg/g WW). Concentrations exceeded the 0.2 μg/g WW aquatic life criterion for mercury in 38% of fish samples. Fish trophic magnification factors (TMFs; range 0.33–3.75) indicated that contaminant accumulation occurred from both water and dietary sources. The combination of analytical approaches applied here provides new insight into contaminant dynamics with conservation implications.