We evaluated the bioaccumulation and transfer of per- and polyfluoroalkyl substances (PFAS) in a stream food web contaminated by a food processing facility. Abiotic (i.e., water, sediment, and foam) and biotic (i.e., algae, aquatic insect larvae and adults, fish, and riparian spiders) matrices were sampled upstream and downstream of the facility’s wastewater outfall. Compared with upstream, PFAS concentrations were 600-fold higher in downstream water (mean ∑₄₀PFAS 3.67 ng mL^–1 ± 0.48 (standard error)) and reflected inputs from the outfall, with 6:2 fluorotelomer sulfonate (6:2 FTS) dominating the PFAS profile. Within the aquatic food web, perfluorooctanesulfonate (PFOS) was the most biomagnified, and 6:2 FTS was the most biodiluted. In contrast, insect-mediated transfer of PFAS to riparian spiders showed trophic enrichment of 6:2 FTS and dilution of PFOS. We observed significant positive associations between phospholipid membrane-water partition coefficient (log K_MW) and perfluoroalkyl carboxylate (PFCA) chain length on bioaccumulation across most biological matrices, demonstrating that these chemical parameters are predictive of PFAS bioaccumulation potential in the field. Our research reveals important differences in aquatic versus terrestrial exposure for certain PFAS and that biological processes (e.g., trophic interactions and metamorphosis) and chemical properties (e.g., chain length, log K_MW, and concentration) control PFAS uptake, bioaccumulation, and transfer in linked freshwater and terrestrial ecosystems.