Temporal patterns in glyphosate and atrazine concentrations were measured weekly by the U.S. Geological Survey (USGS) during the 2013 growing season in 100 small streams in the Midwestern United States. Concentrations also were measured every 2 days at a subset of 8 of the sites, all located in Missouri. Glyphosate was detected more frequently in urban streams than in agricultural streams, and at concentrations similar to those in streams with high agricultural land use in the watershed. In contrast, atrazine was detected more frequently and at higher concentrations in agricultural streams than in urban streams. This data release provides watershed characteristics and 2013 glyphosate and atrazine compound concentrations used in the analysis presented in the journal article “Similarities and differences in temporal fluctuations in glyphosate and atrazine in small Midwestern streams (USA) during the 2013 growing season,” by BJ Mahler, PC Van Metre, TE Burley, KA Loftin, MT Meyer, and LH Nowell, http://dx.doi.org/10.5066/F7SN073J.