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The use of environmental DNA (eDNA) to assess aquatic biodiversity is a growing field with great potential for monitoring and managing threatened species, like freshwater mussel (Unionidae) populations. Freshwater mussels are globally imperiled and serve essential roles in aquatic systems as a food source and as a natural water filter making their management essential for ecosystem health. Unfortunately, mussel populations are often understudied, and challenges exist to accurately and efficiently describe the full suite of species present. Multispecies eDNA approaches may also be more challenging where freshwater mussel populations are most diverse due to ongoing and significant taxonomic restructuring that has been further complicated by molecular phylogenies using mitochondrial genes. For this study, we developed a microfluidic metabarcoding array that targets a wide range of species, from invertebrates to fishes, with an emphasis on detecting unionid mussels known to be present in the Sipsey River, Alabama. We compared mussel species diversity across six sites with well-studied mussel assemblages using eDNA surveys and traditional quadrat surveys in 2016. We examined how factors such as mussel population density, biomass and location in the river substrate impacted our ability to detect certain species; and investigated unexpected eDNA detections through phylogenetic analysis. Our eDNA results for fish and mussel species were broadly consistent with the data from traditional electrofishing and quadrat-based field surveys, although both community eDNA and conventional sampling detected species unique to that method. Our phylogenetic analysis agreed with other studies that treat Pleurobema decisum and P. chattanoogaense as synonymous species; however, they are still listed as unique species in molecular databases which complicates their identity in a metabarcoding assay. We also found that Fusconaia flava and F. cerina are indistinguishable from one another using a portion of the NADH dehydrogenase Subunit 1 (ND1) marker, which may warrant further investigation into whether or not they are synonymous. Our results show that many factors impacted our ability to detect and correctly identify Unionidae mussel species. Here we describe the obstacles we faced, including the murky phylogeny of Unionidae mussels and turbid river conditions, and our development of a potentially impactful freshwater mussel monitoring eDNA assay.