Data are from water samples collected from tributaries of the Great Lakes at three different drainage basin scales, including 1). watershed scale: 8 tributaries of the Great Lakes, 2). subwatershed scale: 5 locations from the greater Milwaukee, Wisconsin area, and 3). small scale: 213 storm sewers and open channel locations in three subwatersheds within the Great Lakes Basin including the Middle Branch of the Clinton River in Macomb County, Michigan (65 sample locations), Red Creek in Monroe County, New York (88 sample locations), and the Kinnickinnic River in Milwaukee County, Wisconsin (60 sample locations). At the watershed- and subwatershed-scale locations, water samples were collected over a 24-hour duration for low-flow periods, and throughout the duration of increased streamflow for runoff-event periods. An individual sample included multiple subsamples that were composited using automatic samplers. At the small-scale locations, discrete grab samples were collected by direct bottle submersion or by peristaltic pump. Water samples were analyzed for absorbance spectra and fluorescence excitation-emission matrices (EEMs), which are presented in this data release. Samples were also analyzed for human-specific viruses, at the watershed- and subwatershed-scale locations only, human- and fecal- indicator bacteria, and dissolved organic carbon (DOC), which are archived in the U.S. Geological Survey National Water Information System (NWIS). These data were used to develop regression models for describing variability of human-associated and fecal indicator bacteria, and an archive of these models is provided. Sample collection, laboratory analyses methods, and a detailed description of the modeling process are described in the associated journal publication: Corsi, S.R., De Cicco, L.A., Hansen, A.M., Lenaker, P.L., Bergamaschi, B.A., Pellerin, B.A., Dila, D.K., Bootsma, M.J., Spencer, S.K., Borchardt, M.A., and McLellan, S.L., 2021, Optical properties of water for prediction of wastewater contamination, human-associated bacteria, and fecal indicator bacteria in surface water at three watershed scales: Environmental Science and Technology, 55, 20, 13770–13782, https://doi.org/10.1021/acs.est.1c02644.