The U.S. Geological Survey is studying the interaction of a contaminated groundwater plume enriched in uranium and other trace elements with water, sediment, and biota along a 3 km reach of the Little Wind River in central Wyoming. The source of the contaminants is from a reclaimed uranium mill site near Riverton, Wyoming. The study is being done in collaboration with the Department of Energy, University of Montana, Northern Arapaho Tribe, and Liverpool John Moores University. This Data Release makes available data collected from June to September, 2016. Data collected during 2016 include: (1) radon, major ion, and trace element concentrations in surface-water, groundwater, and pore-water samples; (2) environmental tracers in groundwater and surface-water samples; (3) seepage rates of shallow groundwater into the Little Wind River; (4) streambed temperature; (5) microbial population composition in streambed biofilms; (6) distribution of uranium in bed sediment, macroalgae, and aquatic insect taxa; and (7) river discharge at three sites along the study reach.