The data presented in this data release includes 11 field/water quality parameters, concentrations of 16 nutrients/anions, dissolved organic carbon, 14 organic contaminants, net estrogenicity concentrations, and 51 inorganic constituents in surface water collected twice from 28 stream sites and collected once from 2 stream sites in the Shenandoah Valley (Virginia and West Virginia, USA), in the summer of 2021 (Table_1_Sites and Table_2_Methods). The estrogenicity, inorganic, and organic chemical characteristics of river waters were analyzed using 19 separate analytical methods at 5 laboratories (Table_2_Methods). Surface water was analyzed for water quality and nutrients (Table_3_WQ_Nutrients_Anions_DOC), toxic contaminants that impact stream and organismal health including a variety of anthropogenic contaminants (i.e., chelating agents, pharmaceuticals, pesticides, and plasticizers) (Table_4_Detected_Toxics_PFAS), perfluoroalkyl and polyfluoroalkyl substances (Table_4_Detected_Toxics_PFAS), and additionally hormone activity (Table_5_Estogenicity), and major and trace elements (Table_6_Inorganics). Quality assurance (QA) field blanks (Table_7_Field_Blanks) and laboratory QA (Table_8_PFAS_Matrix_Spikes and Table_9_AQHAZ_Surrogates) also are presented.