Abstract The ubiquity of microplastics in aquatic ecosystems has raised concerns over their interaction with biota. However, microplastics research on freshwater species, especially mollusks, is still scarce. We, therefore, investigated the factors affecting microplastics ingestion in the freshwater mussel Dreissena polymorpha. Using polystyrene spheres (5, 10, 45, 90 µm), we determined the body burden of microplastics in the mussels in relation to 1) exposure and depuration time, 2) body size, 3) food abundance, and 4) microplastic concentrations. D. polymorpha rapidly ingested microplastics and excreted most particles within 12 h. A few microplastics were retained for up to 1 wk. Smaller individuals had a higher relative body burden of microplastics than larger individuals. The uptake of microplastics was concentration-dependent, whereas an additional food supply (algae) reduced it. We also compared the ingestion of microplastics by D. polymorpha with 2 other freshwater species (Anodonta anatina, Sinanodonta woodiana), highlighting that absolute and relative uptake depends on the species and the size of the mussels. In addition, we determined toxicity of polystyrene fragments (≤63 µm, 6.4–100 000 p mL–1) and diatomite (natural particle, 100 000 p mL–1) in D. polymorpha after 1, 3, 7, and 42 d of exposure, investigating clearance rate, energy reserves, and oxidative stress. Despite ingesting large quantities, exposure to polystyrene fragments only affected the clearance rate of D. polymorpha. Further, results of the microplastic and diatomite exposure did not differ significantly. Therefore, D. polymorpha is unaffected by or can compensate for polystyrene fragment toxicity even at concentrations above current environmental levels. Environ Toxicol Chem 2021;40:2247–2260. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. Abstract Microplastic ingestion by freshwater mussels depends on the exposure time, body size, food availability, and microplastic concentration. Polystyrene fragments (≤63 µm) affected mussels' clearance rate but not mortality, energy reserves, or oxidative stress. PS = polystyrene; MP = microplastics.