To evaluate relationships between different anthropogenic impacts, contaminant occurrence, and fish health, we conducted in situ fish exposures across the Shenandoah River watershed at five sites with different land use. Exposure water was analyzed for over 500 chemical constituents, and organismal, metabolomic, and transcriptomic endpoints were measured in fathead minnows. Adverse reproductive outcomes were observed in fish exposed in the upper watershed at both wastewater treatment plant (WWTP) effluent- and agriculture-impacted sites, including decreased gonadosomatic index and altered secondary sex characteristics. This was accompanied with increased mortality at the site most impacted by agricultural activities. Molecular biomarkers of estrogen exposure were unchanged and consistent with low or non-detectable concentrations of common estrogens, indicating that alternative mechanisms were involved in organismal adverse outcomes. Hepatic metabolomic and transcriptomic profiles were altered in a site-specific manner, consistent with variation in land use and contaminant profiles. Integrated biomarker response data were useful for evaluating mechanistic linkages between contaminants and adverse outcomes, suggesting that reproductive endocrine disruption, altered lipid processes, and immunosuppression may have been involved in these organismal impacts. This study demonstrated linkages between human-impact, contaminant occurrence, and exposure effects in the Shenandoah River watershed and showed increased risk of adverse outcomes in fathead minnows exposed to complex mixtures at sites impacted by municipal wastewater discharges and agricultural practices.