The present survey examines the trophodynamics of a suite of 19 perfluoroalkyl substances (PFASs) in a temperate macrotidal estuary (Gironde, SW France). Across the 147 biota samples (18 taxa) collected, perfluorooctane sulfonate (PFOS), perfluorooctane sulfonamide (FOSA), and C8−C14 perfluoroalkyl carboxylates (PFCAs) were the mostrecurrent analytes. ΣPFASs ranged between 0.66−45 ng per g of wet weight of the whole body. Benthic organisms had relatively high ΣPFASs compared to demersal organisms and displayed specific composition profiles with higher relative abundances of C8 and C9 PFCAs. Trophic magnification factors (TMFs) were determined through the use of linear mixed effect models including censored data, thereby considering data below detection limits as well as the interspecific variability of δ15N and PFAS levels (random effects). TMFs were almost consistently >1 in the benthic food web as well as when considering all data pooled together, providing evidence for the biomagnification of several PFASs in estuarine environments. In addition, in contrast with previous observations, TMFs determined in the estuarine benthic web were found to significantly decrease with increasing chain length for C8−C14 PFCAs and C6−C8 perfluoroalkyl sulfonates. This suggests that PFAS chemical structure might not be necessarily predictive of TMFs, which are also influenced by the trophic web characteristics.