Chemical and microbiological results, quality assurance and quality control, site location, and method information for surface water, bed sediment, and wastewater effluent collected from 34 stream locations across Iowa (United States). Environmental samples were analyzed for a suite of 29 antibiotics, plated on selective media for 15 types of bacteria growth, and DNA was extracted from culture growth and used in downstream polymerase chain reaction (PCR) assays for the detection of 24 antibiotic resistance genes. Environmental field data were collected in-situ for water temperature (°C), specific conductivity (µs/cm), dissolved oxygen (mg/L), pH (standard units), and turbidity (FNU) using a multiparameter sonde and recorded in the National Water Information System (NWIS) (Table S2). Stream discharge (ft3/s) and gage height (ft) values were estimated using available rating curves from adjacent USGS stream gages when available and relative flow was categorized by comparing discharge on the day of sample collection to discharge over the previous three to five years. Samples were analyzed at U.S. Geological Survey laboratories: bacteria enumeration and growth and antibiotic resistance genes at the Michigan Bacteriological Research Laboratory and antibiotic concentrations at the Organic Geochemistry Research Laboratory.