Pools of aquatic plants and benthic invertebrates were collected along with 47 individuals from three cyprinid fish species (Barbus barbus, Gobio gobio, Rutilus rutilus) at a site in the Rhône River (France). Carbon and nitrogen isotopic ratios (δ¹³C and δ¹⁵N) and a wide range of per- and poly-fluorinated chemicals (PFASs) were analysed in all samples. The sum of PFAS concentrations (ΣPFAS) increased from aquatic plants to fish dorsal muscles; molecular profiles were dominated by C9–C13 perfluorocarboxylic acids (PFCAs), while perfluorooctane sulfonate (PFOS) and perfluorooctane sulfonamide (FOSA) were detected in all samples at lower concentrations. ΣPFAS and especially ΣPFCAs were higher in barbels (B. barbus) than in other species, while roaches (R. rutilus) were less contaminated by PFOS than barbels and gudgeons (G. gobio). Gudgeons accumulated significantly higher FOSA concentrations. Young (small) barbels displayed significantly higher PFOS, perfluorononanoic acid (PFNA) and perfluorodecanoic acid (PFDA) concentrations than did large specimens; conversely, perfluorotetradecanoic acid (PFTeDA) concentrations were significantly higher in large barbels. Multiple linear regressions were performed on the whole set of fish samples with size, mass and isotopic ratios as explicative variables, and several single compounds as explained variables. Regardless of the compound, the regressions did not explain much of the contamination variability. However, adding species as a qualitative variable, i.e. performing analyses of covariance (ANCOVAs) improved the fit greatly, while adding sex did not. Diet (i.e. δ¹³C and δ¹⁵N) was the main factor explaining interspecific differences. Biotransformation was assessed by comparing concentration ratios of PFOS or FOSA to their precursors in the food-web compartments. These ratios increased from invertebrates to fish, and differed among fish species, suggesting that biotransformation occurred but was species-specific. Biomagnification factor calculations showed that C11–C13 PFCAs, PFOS and FOSA were apparently biomagnified in barbels and gudgeons.