The widespread detection of antibiotics in the environment is concerning because antibiotics are designed to be effective at small doses. The objective of this work was to quantify the accumulation rates of antibiotics used by humans and animals, spanning several major antibiotic classes (sulfonamides, tetracyclines, fluoroquinolones, and macrolides), in Minnesota lake-sediment cores. Our goal was to determine temporal trends, the major anthropogenic source to these lacustrine systems, and the importance of natural production. A historical record of usage trends for ten human and/or animal-use antibiotics (four sulfonamides, three fluoroquinolones, one macrolide, trimethoprim, and lincomycin) was faithfully captured in the sediment cores. Nine other antibiotics were not detected. Ofloxacin, trimethoprim, sulfapyridine, and sulfamethazine were detected in all of the anthropogenically-impacted studied lakes. Maximum sediment fluxes reached 20.5 ng cm− 2 yr− 1 (concentration 66.1 ng/g) for ofloxacin, 1.2 ng cm− 2 yr− 1 (1.2 ng/g) for trimethoprim, 3.3 ng cm− 2 yr− 1 (11.3 ng/g) for sulfapyridine, and 1.0 ng cm− 2 yr− 1 (1.6 ng/g) for sulfamethazine, respectively. Natural production of lincomycin may have occurred in one lake at fluxes ranging from 0.4 to 1.8 ng cm− 2 yr− 1 (0.1 to 5.8 ng/g). Wastewater effluent appears to be the primary source of antibiotics in the studied lakes, with lesser inputs from agricultural activities.