Wetlands are frequently used in the U.S. for treating nitrate in agricultural runoff. However, numerous other biologically active contaminants, such as antibiotics and nitrification inhibitors, regularly co-occur with nitrate and many can affect the efficiency of nitrate removal. This project evaluated the discrete and combined effects of specific veterinary and human antibiotics and a common nitrification inhibitor (nitrapyrin) on nitrate-N treatment efficiency in saturated sediments and floating treatment wetlands. Sediment and water samples were collected from 3 locations at the USDA Meat Animal Research Facility near Clay Center NE in August 2019 for assessment of background conditions in order to determine the ideal collection site for future microcosm experiments to quantify rates of N-cycling activity in sediments. The sites ranged in historical exposure to inhibitors from (1) little to no relative historical exposure (Control site) (2) moderate relative historical exposure (Grade Control Structure 5), and (3) heavy relative historical exposure to antibiotic runoff from agricultural and cattle grazing land (Reuse Pit (RP)). Laboratory microcosm experiments were then initiated in December 2019 using sediment collected from the historically high exposure site (Reuse Pit) only and an artificial water prepared based on chemistry of the water measured in August 2019. One set of microcosms was amended with an antibiotic mixture (chlortetracycline, sulfadimethoxine, lincomycin, monensin) and another set with nitrapyrin. The targeted range of concentrations for both sets was 0 to 1000 microgram per L. Sediments were exposed to the inhibitors for 3 weeks prior to initiation of the experiments to ensure the microbial communities were affected by the presence of the inhibitors. Anaerobic denitrification and aerobic nitrification potential rates were then quantified after the 3-week exposure period, and samples were collected for microbial community structure and gene abundance. Background sediments from August and December 2019 collections were characterized for potassium chloride extractable nitrogen species, total percent carbon and nitrogen content, microbial community composition, and gene abundance. And background water chemistry was measured from both collections. Data from the N-cycling measurements, molecular analyses, inhibitor concentrations, and background sediment and water characterization are documented in this data release.