Indigenous freshwater mussels (Unionidae) are integral to riverine ecosystems, playing a pivotal role in aquatic food webs and providing ecological services. With populations on the decline worldwide, freshwater mussels are of conservation concern. In this study, we explore the propensity of the invasive Round Goby (Neogobius melanostomus) fish to prey upon indigenous freshwater mussels. First, we conducted lab experiments where Round Gobies were given the opportunity to feed on juvenile unionid mussels and macroinvertebrates, revealing rates and preferences of consumption. Several Round Gobies consumed whole freshwater mussels during these experiments, as confirmed by mussel counts and x-ray images of the fishes. Next, we investigated Round Gobies collected from stream habitats of the French Creek watershed, which is renowned for its unique and rich aquatic biodiversity. We developed a novel DNA metabarcoding method to identify the specific species of mussels consumed by Round Goby and provide a new database of DNA gene sequences for 25 indigenous unionid mussel species. Several of the fishes sampled had consumed indigenous mussels, including the Elktoe (non-endangered), Creeper (non-endangered), Long Solid (state endangered), and Rayed Bean (federally endangered) species. The invasive Round Goby poses a growing threat to unionid mussels, including species of conservation concern. The introduction of the invasive Round Goby to freshwaters of North America is shaping ecosystem transitions within the aquatic critical zone having widespread implications for conservation and management.