Abstract Expansion and intensification of large-scale animal feeding operations (AFOs) in the United States has resulted in concern about environmental contamination and its potential public health impacts. The objective of this investigation was to obtain background data on a broad profile of antimicrobial residues in animal wastes and surface water and groundwater proximal to large-scale swine and poultry operations. The samples were measured for antimicrobial compounds using both radioimmunoassay and liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) techniques. Multiple classes of antimicrobial compounds (commonly at concentrations of >100 μg/l) were detected in swine waste storage lagoons. In addition, multiple classes of antimicrobial compounds were detected in surface and groundwater samples collected proximal to the swine and poultry farms. This information indicates that animal waste used as fertilizer for crops may serve as a source of antimicrobial residues for the environment. Further research is required to determine if the levels of antimicrobials detected in this study are of consequence to human and/or environmental ecosystems. A comparison of the radioimmunoassay and LC/ESI-MS analytical methods documented that radioimmunoassay techniques were only appropriate for measuring residues in animal waste samples likely to contain high levels of antimicrobials. More sensitive LC/ESI-MS techniques are required in environmental samples, where low levels of antimicrobial residues are more likely.