Per- and polyfluoroalkyl substances (PFAS) are environmentally persistent anthropogenic contaminants with bioaccumulation and biomagnification potential. Characterizing PFAS spatial dynamics and biomagnification potential is necessary for risk management. In this study, we analyzed abiotic and biotic samples across the Savannah River Site’s (SRS) watershed for 29 PFAS and 22 metals/metalloids. We detected total PFAS in concentrations ranging from non-detections to 2225.95 ng/L in water, 64.97 ng/g in sediment, and 157.54 ng/g in mosquitofish. We identified strong positive correlations between PFAS and metal concentrations in sediment. To assess PFAS biomagnification, we studied eight species in a stream ecosystem, correlating PFAS concentrations with stable isotope analyses. Total PFAS, long-chain perfluoroalkyl carboxylic acids, perfluorooctane sulfonic acid, and 7:3 fluorotelomer carboxylic acid correlated positively with nitrogen isotope signatures across species, indicating biomagnification potential. Our study characterizes initial environmental and biological PFAS levels on the SRS and provides evidence for PFAS biomagnification within a stream ecosystem.