Dissolved pesticides were measured in weekly water samples from 100 wadeable freshwater streams across eleven states in the Midwestern U.S. during May-August, 2013, as part of the Midwest Stream Quality Assessment study conducted by the U.S. Geological Survey's (USGS) National Water Quality Assessment (NAWQA) Project. Of the 100 stream sites, 12 were urban indicator sites and the remaining 88 sites were located along an agricultural gradient of watershed land use. Twelve depth- and width-integrated water samples were collected at each site within the 14-week study period. Water samples were filtered (0.7 micrometers) and analyzed for 227 pesticide compounds by direct-injection liquid chromatography with tandem mass-spectrometry, and for glyphosate by Enzyme-Linked Immunoassay in a separate analysis. Potential aquatic toxicity was evaluated using the Pesticide Toxicity Index and by comparison to U.S. Environmental Protection Agency aquatic-life benchmarks. This data release provides sampling site locations, method information, summaries of quality-control data, and concentration data for pesticide compounds in environmental weekly water samples, in support of the journal article, “Complex mixtures of dissolved pesticides show potential aquatic toxicity in a synoptic study of Midwestern U.S. streams,” by Nowell, L.H., Moran, P.W., Schmidt, T., Norman, J.E., Nakagaki, N., Shoda, M.E., Mahler, B.J., Van Metre, P.C., Stone, W.W., Sandstrom, M.W., and Hladik, M.L.