This dataset includes the field measurements and laboratory analyses of surface water, seston, fish tissue, and sediment samples collected from Bighorn Lake, within Bighorn Canyon National Recreation area (BICA), during high flow (July 2015) and low flow (August 2016) conditions. The study area includes 7-9 sampling sites that follow a transect spanning the entire length of the reservoir from the Bighorn and Shoshone River inflows to the afterbay and tailwater below Yellowtail dam. Multiple samples were collected at each sampling site and usually included the top 2 cm of sediment at the sediment/water interface, 2 meters above the sediment/water interface, and 2 meters below the reservoir surface. Microbial samples were collected from the water column and sediment samples using sterile techniques and placed on dry ice for laboratory DNA analysis. The samples were analyzed with qPCR and high-throughput DNA sequencing analysis. The qPCR data are archived in the National Center for Biotechnology Information (NCBI) database at: https://www.ncbi.nlm.nih.gov/guide/all/. Water-quality sonde field measurements included specific conductivity, temperature, pH, dissolved oxygen, fluorescent dissolved organic matter, chlorophyll, and turbidity. These field measurements were collected at 16-18 sites at approximately 30 cm increments from the reservoir surface to the reservoir bottom. Discrete water-quality samples that were analyzed for various constituents including selenium and mercury by the U.S. Geological Survey are archived in the National Water Information System (NWIS) database at: https://maps.waterdata.usgs.gov/mapper/index.html. Fish samples were collected during May and June 2015 from throughout Bighorn Lake and in the tailwater below Yellowtail dam and assayed for total mercury, total selenium, and mercury/carbon/nitrogen isotopes.