This dataset includes the field measurements and laboratory analyses of surface water, seston, and sediment collected from Lake Powell, within Glen Canyon National Recreation area (GLCA), during high flow (May-June 2014) and low flow (August 2015) conditions. The study area includes 12-13 sampling sites that follow a transect spanning the entire length of the reservoir from the Colorado River inflow to the Glen Canyon dam, as well as the San Juan River arm, the Escalante River arm and West Canyon. Bed sediment samples were analyzed for mercury speciation, methylmercury production and degradation rates, total reduced sulfur, iron speciation, organic content, and 16S rRNA gene templates as a proxy for microbial abundances. Water samples were collected from 3-5 depths at each site and analyzed for: total mercury (filtered and particulate), methylmercury (filtered and particulate), dissolved organic and inorganic carbon with 13C isotopic ratios, nutrients, anions, cations, trace metals, particulate carbon (with 13C isotopic ratios) and particulate nitrogen (with 15N isotopic ratios). Water quality sonde (EXO) field measurements included specific conductivity, temperature, pH, dissolved oxygen, fluorescent dissolved organic matter, chlorophyll, and turbidity. Fish samples were also collected during November 2014 from Good Hope Bay (upper reservoir), Wahweap Bay (lower reservoir), and the San Juan arm and assayed for total mercury for comparison with previous striped bass samples collected by the state of Utah. There are nine files (*.csv) in this dataset: 1) data dictionary ; 2) sediment data; 3) water data; 4) seston data; 5) fish data; 6) EXO main channel profile data ; 7) EXO off channel profile data; 8) quality assurance data; and 9) molecular data.