A pilot study among farming households in eastern Iowa was conducted to assess human exposure to neonicotinoids (NEOs). The study was in a region with intense crop and livestock production and where groundwater is vulnerable to surface-applied contaminants. In addition to paired outdoor (hydrant) water and indoor (tap) water samples from private wells, urine samples were collected from 47 adult male pesticide applicators along with the completions of dietary and occupational surveys. Estimated Daily Intake (EDI) were then calculated to examine exposures for different aged family members. NEOs were detected in 53% of outdoor and 55% of indoor samples, with two or more NEOs in 13% of samples. Clothianidin was the most frequently detected NEO in water samples. Human exposure was ubiquitous in urine samples. A median of 10 different NEOs and/or metabolites were detected in urine, with clothianidin, nitenpyram, thiamethoxam, 6-chloronicotinic acid, and thiacloprid amide detected in every urine samples analyzed. Dinotefuran, imidaclothiz, acetamiprid-N-desmethyl, and N-desmethyl thiamethoxam were found in ≥70% of urine samples. Observed water intake for study participants and EDIs were below the chronic reference doses (CRfD) and acceptable daily intake (ADI) standards for all NEOs indicating minimal risk from ingestion of tap water. The study results indicate that while the consumption of private well tap water provides a human exposure pathway, the companion urine results provide evidence that diet and/or other exposure pathways (e.g., occupational, house dust) may contribute to exposure more than water contamination. Further biomonitoring research is needed to better understand the scale of human exposure from different sources.