Freshwater mussel conservation efforts by many federal and state agencies have increased in recent years. This has led to a greater number of stream surveys, in which mussel die-offs involving high numbers of dead and moribund animals are being observed and reported with greater frequency. Typically, die-offs have been incidentally observed while research was being done for other purposes, therefore, accurate mortality data have been difficult to obtain. Specifically, seasonal die-offs were noted in localized areas of the Clinch and Holston Rivers, Virginia, and to lesser degrees, in neighboring rivers in this geographic region, including southeast Virginia. The observed mussel species affected were primarily the slabside pearlymussel (Lexingtonia dolabelloides) and to lesser extents, the pheasantshell (Actinonaias pectorosa), rainbow mussel (Villosa iris), and the endangered shiny pigtoe (Fusconaia cor). To determine if a bacterial pathogen might be involved in these recurring mussel die-offs, this study examined characteristics of the indigenous microbiota (bacteria) from healthy mussels from sites on the Clinch and Holston Rivers where die-offs were previously observed. These baseline data will allow for recognition of bacterial pathogens in future mussel die-offs. Means for total bacteria from soft tissues ranged from 1.77 × 10⁵ to 3.55 × 10⁶ cfu/g; whereas, the range in means from fluids was 2.92 × 10⁴ to 8.60 × 10⁵ cfu/mL. A diverse microbiota were recovered, including species that are common in freshwater aquatic environments. The most common bacterial groups recovered were motile Aeromonas spp. and nonfermenting bacteria. Flavobacterium columnare, a pathogen to cool- and warm-water fishes was recovered from one specimen, a Villosa iris from the Clinch River.