An in situ bioassay approach was used to determine whether aquatic contaminant stressors in a large Atlantic river ecosystem affect the survival of 3 fish species: the largemouth bass (Micropterus salmoides, juveniles), the fathead minnow (Pimephales promelas, adults), and the robust redhorse (Moxostoma robustum, juveniles). Hatchery-propagated fish were placed into cages to assess site-specific survival in the Yadkin-Pee Dee River of North Carolina and South Carolina, USA. Contaminants were measured in caged fish and sediment and surface water at each site. No apparent longitudinal trends in fish survival were detected, and contaminant concentrations varied among sites. Juvenile largemouth bass and robust redhorse did not survive past 13 and 23 d, with corresponding Kaplan-Meier median survival estimates of 9.7 and 12.1 d, respectively. Survival of adult fathead minnows deployed in cages alongside the juvenile fish averaged 43% at the end of the 28-d exposure, with a 22-d median survival estimate. The intersex condition, an indicator of endocrine disruption, was not observed in any adult fathead minnow. Contaminant accumulation in surviving fathead minnows was apparent, with highest accumulated concentrations of polychlorinated biphenyls (34.6-93.4 ng/g dry wt), organochlorine pesticides (19.9-66.1 ng/g dry wt), and mercury (0.17-0.63 μg/g dry wt). Contaminants and other water quality stressors in this river system appear to detrimentally impact juvenile fish survival, with presumed effects at the fish assemblage and community levels. Environ Toxicol Chem 2021;40:2206-2219. © 2021 SETAC.