This dataset includes field hydrologic measurements and laboratory analyses of surface and pore waters, sediments, benthic plants/biofilms, and biota along the Middle Snake River upgradient of the Hells Canyon Complex. The study region for this work focuses on a section of the Snake River heavily utilized for agriculture, with complex systems of irrigation diversion and return drainage, spanning 164 km above the Hells Canyon Complex (Idaho, Oregon). The goal of this study was to identify potential loading sources of methylmercury from the upgradient Snake River into the reservoir complex, with a focus on the tributaries, irrigation return drains, and riparian zones adjacent to agricultural lands along the river. To this end, 16 locations along the Snake River between River Miles 448 and 346 were sampled over a four-day period, July 11 – 14, 2022. At each sampling site, pore water and sediment cores were collected in the shallow riparian zones along the edge of the river, surface waters were collected from cross-sections of the river, river flow and velocity were measured, submerged macrophytes and associated biofilm was collected, and biota (snails, amphipods, clams, periphyton, dragonfly larvae) were sampled from shallow riparian margins using a boat-mounted benthic suction dredge. Concurrently, water samples were collected from the six major tributaries that enter the Snake River in the study reach (Succor Creek, Owyhee River, Boise River, Malheur River, Payette River, Weiser River) and from six irrigation return drains that were actively discharging to the Snake River. Data from additional irrigation return drains to the study reach, sampled in June 2021 and May 2022, are also included. All water samples (surface and pore water) were processed and analyzed for filter-passing total mercury and methylmercury, dissolved organic carbon (DOC), dissolved organic matter (DOM) composition (UV-vis absorption and fluorescence), inorganic anions (chloride, nitrate, sulfate), and filter acidified metals (iron, manganese). In addition, the surface waters were analyzed for total suspended solids (TSS), particulate total and methylmercury, particulate organic carbon (POC) and stable isotopic composition, particulate nitrogen (PN) content and stable isotopic composition, and particulate metals (iron, manganese); and the pore waters were analyzed for inorganic sulfide (S(-II)). Sediment, submerged macrophytes and biofilms, and biota samples were dried, ground, and analyzed for total and methylmercury. Loss on ignition was also measured on the sediment samples. This data release includes eight metadata and data tables in machine readable format (*.csv). All data columns in tables 3-8 are defined in Table 2. Reference Table 2 for associated units, abbreviation definitions, laboratory information, and method citations. Table_1_Site_Descriptions.csv - site descriptions Table_2_Analysis_Descriptions.csv - data dictionary defining column headings in Tables 3-8 Table_3_Water_SW.csv - surface water chemistry data Table_4_Water_PW.csv - pore water chemistry data Table_5_Sediment.csv - sediment chemistry data Table_6_Macrophytes_and_Biofilms.csv - submerged macrophyte and biofilm data Table_7_Hydrology.csv - hydrologic data Table_8_Biota.csv - biota data