Dissolved pesticides were measured in weekly water samples from 100 wadeable 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 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 (ELISA) 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. A mesocosm experiment was conducted to investigate the effects of the insecticide imidacloprid on natural stream communities transported to experimental streams in the laboratory, where they were exposed to imidacloprid under controlled conditions. This Data Release provides sampling site locations and watershed characteristics, pesticide analyte information, agricultural pesticide use data, summaries of quality control data, concentration data for pesticide compounds in environmental weekly water samples, and a summary of invertebrate metrics and imidacloprid concentration data from the mesocosm study, 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.