<p>Per- and polyfluoroalkyl substances (PFAS) present a significant environmental issue due to their widespread occurrence and potential toxicity to exposed organisms. Our study investigated PFAS concentrations present in a range of freshwater biota exposed to wastewater discharge and urban stormwater runoff from creeks and rivers in Victoria, Australia. We also assessed bioaccumulation factors (BAFs) in fish, and conducted a human health risk assessment. We sampled 18 sites located along seven waterways (4 effluent-receiving, 1 urban stormwater and 2 reference waterways) with three sampling locations at each of the polluted waterways: at the discharge point, and upstream and downstream of discharge. We analysed freshwater (<i>n</i> = 56), crustacea (<i>n</i> = 18), and fish (<i>n</i> = 242 from 12 species) samples for 32 PFAS. Our study shows that the concentrations and range of PFAS present were highest in samples from the urban stormwater waterway (mean ∑₃₂PFAS water concentration = 0.12&#xa0;µg/L), followed by the wastewater-exposed waterways (mean ∑₃₂PFAS = 0.027&#xa0;µg/L), and then the control reference waterways (mean ∑₃₂PFAS = 0.0043&#xa0;µg/L). The higher concentrations found in the urban waterway suggests that stormwater may be a greater source of PFAS pollution than wastewater discharges. Additionally, the highest BAFs were for PFOS, PFDA, and PFHxS, and occurred in fish with a carnivorous diet. Lastly, consumption of three portions of fish per week caught from the urban stormwater and several effluent-receiving waterways could result in exceedances of health-based guidance values for PFOS in children.</p>