The Southeastern USA is currently experiencing a period of rapid human population growth that is having profound effects on the region’s unique aquatic biota. We used repeated surveys, experiments, and water quality measurements to assess the influences of municipal discharges on water quality, habitat conditions, and freshwater mussel populations in a Piedmont stream. Chewacla Creek, located in east-central Alabama, receives wastewater discharge from the City of Auburn via Parkerson Mill Creek. From 1999-2006 we monitored freshwater mussel populations and physicochemical habitat parameters in Chewacla and Parkerson Mill creeks. Surveys revealed that mussel numbers decreased dramatically immediately downstream of Parkerson Mill Creek. Subsequently, we conducted sentinel trials using adult mussels (Villosa lienosa) in Chewacla Creek and Parkerson Mill Creek. Sentinel mussels were placed in cages at three locations downstream of the effluent discharge point and at one upstream site (control). Sentinel mussels exposed to wastewater discharge exhibited dramatically higher mortality compared to control animals. Combined impacts of wastewater discharges and streambed destabilization are exacerbating fragmentation of Chewacla Creek mussel populations. Both survey and experimental data demonstrate that tributary sub-catchments negatively impact downstream mussels. Moreover, our data indicate that protection of sensitive aquatic taxa necessitates management of the entire watershed.