Identifying and conserving functioning headwater ecosystems is essential for ensuring the structure and function of natural ecosystem processes. The Passage Creek watershed (PCW) in Virginia is an upland tributary system of the North Fork Shenandoah River, which is found within the Potomac River basin. The PCW appears to maintain an array of terrestrial and fluvial habitats now uncommon in many adjacent watersheds because of human disturbance. We assessed the biotic condition of the PCW by sampling the fish, mollusk, and salamander assemblages throughout the watershed. We observed 29 fish, 9 salamander, and 4 aquatic mollusk species representing a variety of life histories and functional groups. Furthermore, we found that due to spatial differences in abundance and species richness, each assemblage offered unique insight into the condition of the PCW. The fish assemblage was indicative of those found in least disturbed areas within the Potomac basin, while salamander abundance and richness indicated areas of habitat degradation. Though we observed only one mussel species, the presence of native mussels suggested the PCW has maintained sufficient ecological condition to support long-lived animals potentially sensitive to low-level, additive, and compounding long-term disturbances, while neighboring watershed assemblages have collapsed. Given the relatively high species richness found within PCW, this watershed may be pivotal to the overall persistence of aquatic species in the Potomac basin and should receive high priority for future conservation efforts.