PFAS contamination of urban waters is widespread but understanding the biological impact of its accumulation is limited to humans and common ecotoxicological model organisms. Here, we combine PFAS exposure and bioaccumulation patterns with whole organism responses and omics-based ecosurveillance methods to investigate the impacts of PFAS on a wetland top predator, the tiger snake (Notechis scutatus). Tiger snakes (18 male and 17 female) were collected from four wetlands with varying PFAS chemical profiles and concentrations in Perth, Western Australia. Tiger snake livers were tested for 28 known PFAS compounds, and Σ28PFAS in liver tissues ranged between 322 ± 193 µg/kg at the most contaminated site to 1.31 ± 0.86 µg/kg at the least contaminated site. The dominant PFAS compound detected in liver tissues was PFOS. Lower body condition was associated with higher liver PFAS, and male snakes showed signs of high bioaccumulation whereas females showed signs of maternal offloading. Biochemical profiles of snake muscle, fat (adipose tissue), and gonads were analysed using a combination of liquid chromatography triple quadrupole (QqQ) and quadrupole time-of-flight (QToF) mass spectrometry methodologies. Elevated PFAS was shown to enrich energy production and maintenance pathways in the muscle, and had loose associations with energy-related lipids and bile acids in the fat tissue, and lipids associated with cellular genesis and spermatogenesis in the gonads. These data demonstrate the bioavailability of urban wetland PFAS in higher-order reptilian predators and indicate a negative impact on snake health and metabolic processes. This research contributes to our understanding of the impact of PFAS residue on wildlife health which can be used for improved risk management and regulation. Thus, expanding the use of omics-based ecosurveillance tools for informing mechanistic toxicology.