This data release presents chemical and biological results from investigations of water quality, fish endocrine disruption, and emergent insects in the Shenandoah River Watershed (Virginia and West Virginia, USA) conducted during 2014, 2015, and 2016. Multiple sampling campaigns were conducted at sites located throughout the Shenandoah River Watershed (Table 1). The complex inorganic and organic chemical characteristics of river waters and wastewater treatment plant effluents were characterized using 21 separate analytical methods at 7 laboratories (Tables 2, 3, and 4). To assess the relations between water composition and fish endocrine disruption, 21-day mobile laboratory adult fathead minnow exposure experiments with water quality and biological characterization (water collected on day 0, 7, 14, and 21; fish collected on day 0, 7, and 21) were conducted at 4 locations from August 12 to September 1, 2014, 3 locations from May 26 to June 16, 2015, and 5 locations from July 27 to August 17, 2016 (Tables 1 and 5). To assess changes in water chemistry resulting from point and non-point sources along the flow path, targeted stream-reach sampling for water quality characterization was conducted at 14 locations from August 25 to August 27, 2015 (Tables 1 and 6). Temporal variability was assessed by time-series sampling for water quality characterization at 2 locations associated with wild fish collection from April 4 to June 14, 2016 (Tables 1 and 7). Spatial variability across the watershed was assessed by landscape-directed sampling for water-quality characterization with biological assessment at 18 locations from August 9 to August 16, 2016, and 17 locations from October 17 to October 18, 2016 (Tables 1 and 8). Biological results for the 21-day mobile-laboratory adult fathead minnow exposure experiments are presented in Table 9. Biological results for the wild smallmouth bass and white sucker sampling on April 12, 2016 are presented in Table 10. Biological results for the 96-hour larval fathead minnow exposures from July, August, and October 2016 are presented in Table 11. Results for tissue analysis of pesticides and disinfection byproducts in adult fathead minnows collected at day 21 of the August to September 2014 exposure experiments are presented in Table 12. Emergent aquatic insects, terrestrial insects, and spider sampling was conducted at 4 locations from May 21 to May 25, 2015 (Tables 13, 14, and 15) and tissue samples analyzed for pesticides (Tables 16 and 17).