Complex chemical mixtures have been widely reported in larger streams but relatively little work has been done to characterize them and assess their potential effects in headwaterstreams. In 2014, the United States Geological Survey (USGS) sampled 54 Piedmont streams over ten weeks and measured 475 unique organic compounds using five analytical methods. Maximum and median exposure conditions were evaluated in relation to watershed characteristics and for potential biological effects using multiple lines of evidence. Results demonstrate that mixed-contaminant exposures are ubiquitous and varied in sampled headwater streams. Approximately 56% (264) of the 475 compounds were detected at least once across all sites. Cumulative maximum concentrations ranged 1,922–162,346 ng L⁻¹ per site. Chemical occurrence significantly correlated to urban land use but was not related to presence/absence of wastewater treatment facility discharges. Designed bioactive chemicals represent about 2/3rd of chemicals detected, notably pharmaceuticals and pesticides, qualitative evidence for possible adverse biological effects. Comparative Toxicogenomics Database chemical-gene associations applied to maximum exposure conditions indicate >12,000 and 2,900 potential gene targets were predicted at least once across all sites for fish and invertebrates, respectively. Analysis of cumulative exposure-activity ratios provided additional evidence that, at a minimum, transient exposures with high probability of molecular effects to vertebrates were common. Finally, cumulative detections and concentrations correlated inversely with invertebrate metrics from in-stream surveys. The results demonstrate widespread instream exposure to extensive contaminant mixtures and compelling multiple lines of evidence for adverse effects on aquatic communities.