Organic chemicals from industrial, agricultural, and residential activities can enter surface waters through regulated and unregulated discharges, combined sewer overflows, stormwater runoff, accidental spills, and leaking septic-conveyance systems on a daily basis. The impact of point and nonpoint contaminant sources can result in adverse biological effects for organisms living in or near surface waters. Assessing the adverse or toxic effects that may result when exposure occurs is complicated by the fact that many commonly used chemicals lack toxicity information or water quality standards. To address these challenges, an exposure-activity ratio (EAR) screening approach was used to prioritize environmental chemistry data in a West Virginia watershed (Wolf Creek). Wolf Creek is a drinking water source and recreation resource with documented water quality impacts from point and nonpoint sources. The EAR screening approach uses high-throughput screening (HTS) data from ToxCast as a method of integrating environmental chemical occurrence and biological effects data. Using water quality schedule 4433, which targets 69 organic waste compounds typically found in domestic and industrial wastewater, chemicals were screened for potential adverse biological affects at multiple sites in the Wolf Creek watershed. Cumulative EAR mixture values were greatest at Sites 2 and 3, where bisphenol A (BPA) and pentachlorophenol exhibited maximum EAR values of 0.05 and 0.002, respectively. Site 2 is downstream of an unconventional oil and gas (UOG) wastewater disposal facility with documented water quality impacts. Low-level organic contaminants were found at all sample sites in Wolf Creek, except Site 10, where Wolf Creek enters the New River. The application of an EAR screening approach allowed our study to extend beyond traditional environmental monitoring methods to identify multiple sites and chemicals that warrant further investigation.