Per- and polyfluoroalkyl substances (PFAS) are a group of synthetic chemicals detected throughout the environment. To better understand the distribution of PFAS in an aquatic system (the Laurentian Great Lakes), stable isotope enrichment (δ13C and δ15N), fatty acid (FA) profiles, and PFAS were measured in various species from the Lake Ontario (LO) aquatic food web. Sampled organisms included top predator fish, prey fish, and benthic and pelagic macroinvertebrates. The trophic level of each species in the LO food web was determined using δ15N, and FA concentrations (range: <1–139 mg/g wet weight (ww)). The individual PFAS concentrations in the LO food web were ~1.5 to 5 times lower than previously reported. The highest PFAS concentrations were observed in deepwater sculpin (Myoxocephalus thompsonii, 150 ± 35.7 ng/g ww) suggesting a potential source of PFAS from the offshore benthic zone or sediment. The concentration of PFOS and long chain (C9-C14) perfluoroalkyl carboxylic acids (PFCAs) were significantly higher than short chain PFAS indicating the significant impact of hydrophobicity on the bioaccumulation of PFAS in organisms from the food web. However, high molecular weight PFCAs (>C8) did not exhibit increasing biomagnification factors (BMFs) and trophic magnification factors (TMFs) with log Kow, suggesting hydrophobicity does not govern the movement of PFAS from low to high trophic levels in the LO food web.