Reuse of municipal and industrial wastewater treatment plant (WWTP) effluent is used to augment freshwater supplies globally. The Shenandoah River Watershed (U.S.A.) was selected to conduct on-site exposure experiments to assess endocrine disrupting characteristics of different source waters. This investigation integrates WWTP wastewater reuse modeling, hydrological and chemical characterization, and in vivo endocrine disruption bioassessment to assess contaminant sources, exposure pathways, and biological effects. The percentage of accumulated WWTP effluent in each river reach (ACCWW%) was used to predict environmental concentrations for consumer product chemicals (boron), pharmaceutical compounds (carbamazepine), and steroidal estrogens (estrone, 17-β-estradiol, estriol, and 17-α-ethinylestradiol). Fish endocrine disruption was evaluated using vitellogenin induction in adult male or larval fathead minnows. Water samples were analyzed for >500 inorganic and organic constituents to characterize the complex contaminant mixtures. Municipal ACCWW% at drinking water treatment plant surface water intakes ranged from <0.01 to 2.0% under mean-annual streamflow and up to 4.5% under mean-August streamflow. Measured and predicted environmental concentrations resulted in 17-β-estradiol equivalency quotients ranging from 0.002 to 5.0 ng L–1 indicating low-to-moderate risk of fish endocrine disruption. Results from the fish exposure experiments showed low (0.5- to 3.2-fold) vitellogenin induction in adult males.