Widespread contamination of per- and polyfluorinated alkyl substances (PFAS) from wastewater effluents may pose risks to fish and human health associated with bioaccumulation. Greater understanding of the bioaccumulation of PFAS in fish (and crustacea) is crucial for characterising toxicity, assessing risks to human consumption, and setting guidelines. The overall aim of this study was to determine concentrations of PFAS in fish from seven streams in Victoria, Australia. Specifically, we aimed to: (1) assess concentrations and bioaccumulation of PFAS in fish relative to wastewater discharge; (2) determine differences in PFAS concentrations between locations, streams, and species. Fish were sampled at 18 sites from seven streams: four receiving wastewater effluent, one urban catchment and two reference streams. At least 20 individual fish were collected from each site, comprising 360 fish in total. Fish sampled included native (blackfish, long-finned eels, shrimp, crayfish) and invasive (carp, redfin perch, roach) species. Water concentrations were assessed using passive samplers and grab samples. The water data showed that out of 30 PFAS compounds analysed, the four highest concentrations included PFOA (<0.001 to 0.031 µg/L), PFOS (<0.001–0.03 µg/L), PFHxA (<0.01–0.03 ug/L), and PFHxS (<0.001–0.014 µg/L). In the most urbanised stream, PFAS concentrations were significantly higher (pseudo-F3,24=18.2, p<0.001) with mean concentrations of PFOS (0.02 µg/L) close to four times higher than in other peri-urban streams (0.005 µg/L), and 10 times higher than in reference streams (0.002 µg/L). Out of 30 PFAS compounds, 8 were detected in fish samples (muscle). Highest PFOS concentrations were found in redfin perch, followed by carp, and short-finned eel (PFOS <0.5–160 µg/kg). Concentrations in fish aligned with those observed in water. Bioaccumulation factors (BAF log10) for PFOS ranged from 2.45 (shrimp) to 5 (roach). The study data highlights that species-specific bioaccumulation rates of PFAS should be considered when developing human and ecological risk assessment.