Freshwater mussels (Mollusca: Unionidae) play a crucial role in freshwater river environments where they live in multi-species aggregations and often serve as long-lived benthic ecosystem engineers. Many of these species are imperiled and it is imperative that we understand their basic needs to aid in the reestablishment and maintenance of mussel beds in rivers. To expand our knowledge of the diet of these organisms, ten species of mussel were introduced into enclosed systems in three experiments. In the first, five species of relatively common mussels were incubated in water from the Clinch River (Virginia, USA) and in the second, water from a manmade pond at the Commonwealth of Virginia’s Aquatic Wildlife Conservation Center in Marion, VA. In the third experiment, five endangered species were incubated in water from the AWCC pond. Quantitative PCR and eDNA metabarcoding were used to determine which planktonic microbes were present before and after the introduction of mussels into each experimental system. It was found that all ten species preferentially consumed microeukaryotes over bacteria. Most microeukaryotic taxa, including Stramenopiles and Alveolates were quickly consumed by all ten mussel species. We also found that they consumed fungi but not as quickly as the microalgae, and that only two species of mussels, Ortmanniana pectorosa and Medionidus conradicus, may have consumed bacteria. Our results provide evidence that siphon feeding Unionid mussels can select preferred microbes from mixed plankton, and mussel species may exhibit dietary niche differentiation.