These datasets are one component of the multistressor studies conducted in Midwest streams in 2013 (MSQA) and in Southeast streams in 2014 (SESQA) by the U.S. Geological Survey National Water Quality Assessment Project. High-frequency small-volume autosamplers were deployed at 7 sites each in MSQA and SESQA that collected daily and weekly composite water samples, which were analyzed for 225 pesticides and pesticide degradates. Five of the MSQA autosampler sites were in agricultural watersheds and two in urban watersheds, whereas all seven SESQA autosampler sites were in urban watersheds. The daily and weekly composite samples were compared with results from traditional discrete water samples collected weekly at the sites. Mixtures of pesticides were present in most samples and the Pesticide Toxicity Index (PTI) and acute invertebrate benchmarks were used to evaluate the potential for acute invertebrate toxicity of mixtures. This Data Release provides concentration data for pesticide compounds in environmental weekly and daily composite water samples, pesticide analyte information, summaries of quality control data, and PTI scores, in support of the journal article “Daily-composite stream samples reveal highly complex pesticide occurrence and potential toxicity to aquatic life,” by Norman, J.E., Mahler, B.J., Nowell, L.H., Van Metre, P.C., Sandstrom, M.W., Corbin, M.A., Qian, Y., Pankow, J.F., Luo, W., Fitzgerald, N.B., Asher, W.E., and McWhirter, K.J.