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Microplastics are widespread in the environment, including in the bodies of freshwater fish, with their concentrations influenced by factors such as proximity to point sources, such as wastewater treatment plants (WWTPs), and trophic level. However, few studies have simultaneously assessed the combined effects of these factors on microplastic abundance in urban stream fish. To do so, we measured microplastics and assessed trophic level via N stable isotope (δ15N) content in 6 species of small-bodied fishes (species = Lepomis macrochirus Rafinesque, 1819, Neogobius melanostomus [Pallas, 1814], Fundulus notatus [Rafinesque, 1820], Pimephales notatus [Rafinesque, 1820], Notemigonus crysoleucas [Mitchill, 1814], and Dorosoma cepedianum [Lesueur, 1818]) collected upstream, at, and downstream of a large WWTP in Chicago, Illinois, USA. Additionally, we analyzed stomach contents for 2 of these species (L. macrochirus and N. melanostomus). Four of the six species exhibited δ15N enrichment at and downstream of the WWTP, indicating prolonged residence times at the study sites (i.e., several weeks). Stomach contents of the 2 species measured supported this pattern, showing high chironomid consumption at the WWTP and variable stomach contents elsewhere. For microplastics, 1 species had higher concentrations near the WWTP, but microplastic concentrations did not differ among locations in the other 5 species. We found no evidence of a relationship between δ15N enrichment and microplastic concentration. Overall, the stable isotope and stomach content results suggest a strong relationship of WWTP effluent with fish diet but not with microplastic concentrations in fish. The results suggest that microplastic concentrations in fish is are shaped by interacting, species-specific factors including behavior (i.e., movement and foraging) and physiology (i.e., egestion rates and feeding mechanisms), in addition to proximity to point sources. Our study highlights the complexity of microplastic infiltration into food webs and underscores the need for further research to disentangle the drivers of microplastic accumulation in aquatic organisms.