A multimillion-dollar salmonid fishery in Lake Michigan is enabled by abundant prey fish that serve as a key link between basal resources and top predators. This role also makes prey fish an important conduit for per- and polyfluoroalkyl substances (PFAS) in the food web. We quantified 21 PFAS in six prey fish species (alewife, bloater, round goby, deepwater sculpin, slimy sculpin, and gizzard shad) to understand how habitat use, feeding guild, and ontogeny influence PFAS accumulation in the critical middle of the Lake Michigan food web. Fish were collected from seven harbors encircling the lake during the summers of 2020 and 2021. PFAS were detected in all fish (N = 301), with species-specific concentrations ranging from 3.22 ± 0.8 (gizzard shad; mean ± SE) to 100.9 ± 11 ng/g wet weight (slimy sculpin); upper-end concentrations were notably high relative to top predators studied previously in the same region (Lake Michigan, Great Lakes, North America). Clustered isotopically (δ15N and δ13C), benthic species associated with fine substrates (deepwater and slimy sculpins; 45.7 ± 2.63) had higher PFAS than those found on coarse substrates (round goby; 3.71 ± 0.35), suggesting that fine sediments are an important PFAS reservoir and exposure pathway for organisms. Pelagic zooplanktivores combined (alewife and bloater; 6.18 ± 0.07) had intermediate PFAS concentrations, whereas the filter-feeding planktivore gizzard shad exhibited the lowest overall PFAS levels. Carbon stable isotopes indicated narrow dietary niches for most species, with broader niches for gizzard shad and round goby. However, broader diets were not associated with higher PFAS loads in these species. Ontogenetic patterns appeared to influence PFAS uptake; PFOS, the most frequently detected PFAS, increased with age in deepwater sculpin, decreased with age in bloater, and showed no pattern in alewife, likely reflecting species-specific habitat shifts and benthic–pelagic coupling. Overall, PFAS accumulation was strongly influenced by species ecology, highlighting how ecological traits in the food web can influence contaminant transfer to higher trophic levels.