This study examined the release of per- and poly fluoroalkyl substances (PFAS) into an urban freshwater stream and sediments due to a large-scale industrial fire in Melbourne, Australia. The fire occurred at an industrial warehouse which was illegally storing excessive amounts of unknown chemicals and industrial waste. The runoff of the waste and used firefighting foam was contained in the adjacent freshwater creeks. The concentration of 50 PFAS in freshwater samples and 16 PFAS in sediment samples were determined by HPLC-MS/MS. Of the fifty targeted PFAS in the freshwater samples, fluorotelomer sulfonates including (6:2 fluorotelomer sulfonamide alkylbetaine (6:2 FTAB or 6:2 FTSA-PrB), Perfluorocarboxylates (PFCAs: C4-C12), Perfluoroalkane sulfonates (PFSAs: C4-C8) and perfluoroalkanesulfonyl fluorides including perfluorobutane sulfonamide (FBSA) were the most elevated PFAS. There were clear differences in Σ50 PFAS mean concentrations detected on the day of the fire (3843 ng/L) relative to one month post fire (352 ng/L), indicating the effect of the fire on freshwater system. The concentration of Σ50 PFAS at the point source was 3400 ± 324 ng/L on the day of the ignition and decreased significantly to 93 ± 14 ng/L in the second campaign. This is the first study to document 6:2 FTAB and perfluorohexane sulfonamide (FHxSA), perfluoro-4-methoxybutanoic acid (PFMBA), perfluoro-3-methoxypropanoic acid (PFMPA), Hexafluoropropylene oxide dimer acid (HFPO-DA), Nonafluoro-3,6-dioxaheptanoic acid (NFDHA) and Perfluoro(2-ethoxyethane) sulfonic acid (PFEESA) in Australian surface waters. Only three PFAS were detected in the sediment samples which collected from five sites in the second campaign which could be negligible based on the detection. In the meanwhile, the concentration of PFAS were also compared to the available guideline values in Australia for freshwater, recreational water, and sediments. The analysed water samples indicated that PFOS contamination level might have a short-term risk to the aquatic organisms.