Anthropogenically modified microparticles including microplastics are present in municipal wastewater treatment plant (WWTP) effluents; however, it is unclear whether biotic exposures are elevated downstream of these outfalls. In the fall of 2019, the present study examined whether microparticle levels in resident fish, environmental samples, and caged organisms were elevated near the Waterloo and Kitchener WWTP outfalls along the Grand River, Ontario, Canada. Wild rainbow darters (Etheostoma caeruleum) were collected from a total of 10 sites upstream and downstream of both WWTPs, along with surface water and sediment samples to assess spatial patterns over an approximately 70‐km river stretch. Amphipods (Hyalella azteca), fluted‐shell mussels (Lasmigona costata), and rainbow trout (Oncorhynchus mykiss) were also caged upstream and downstream of one WWTP for 14 or 28 days. Whole amphipods, fish digestive tracts, and mussel tissues (hemolymph, digestive glands, gills) were digested with potassium hydroxide, whereas environmental samples were processed using filtration and density separation. Visual identification, measurement, and chemical confirmation (subset only) of microparticles were completed. Elevated abiotic microparticles were found at several upstream reference sites as well as at one or both wastewater‐impacted sites. Microparticles in amphipods, all mussel tissues, and wild fish did not show patterns indicative of increased exposures downstream of effluent discharges. In contrast, elevated microparticle counts were found in trout caged directly downstream of the outfall. Across all samples, cellulose fibers (mainly blue and clear colors) were the most common. Overall, results suggest little influence of WWTP effluents on microparticles in biota but rather a ubiquitous presence across most sites that indicates the importance of other point and nonpoint sources to this system. Environ Toxicol Chem 2024;00:1–15. © 2024 His Majesty the King in Right of Canada and The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. Reproduced with the permission of the Minister of Environment and Climate Change Canada.