Antimicrobial resistance is a growing public health problem that requires an integrated approach among human, agricultural, and environmental sectors. However, few studies address all three components simultaneously. We investigated the occurrence of five antibiotic resistance genes (ARGs) and the class 1 integron gene (intI1) in private wells drawing water from a vulnerable aquifer influenced by residential septic systems and land-applied dairy manure. Samples (n = 138) were collected across four seasons from a randomized sample of private wells in Kewaunee County, Wisconsin. Measurements of ARGs and intI1 were related to microbial source tracking (MST) markers specific to human and bovine feces; they were also related to 54 risk factors for contamination representing land use, rainfall, hydrogeology, and well construction. ARGs and intI1 occurred in 5–40% of samples depending on target. Detection frequencies for ARGs and intI1 were lowest in the absence of human and bovine MST markers (1-30%), highest when co-occurring with human and bovine markers together (11-78%), and intermediate when co-occurring with just one type of MST marker (4-46%). Gene targets were associated with septic system density more often than agricultural land, potentially because of the variable presence of manure on the landscape. Determining ARG prevalence in a rural setting with mixed land use allowed an assessment of the relative contribution of human and bovine fecal sources. Because fecal sources co-occurred with ARGs at similar rates, interventions intended to reduce ARG occurrence may be most effective if both sources are considered.