Private wells supply drinking water for many households, and their contamination by fecal microbes presents a risk of acute gastrointestinal illness (AGI). Risk is thought to vary by contaminating fecal source, but specific associations with fecal source are unknown for most private wells in the United States. This study characterizes AGI risk in Grant, Iowa, and Lafayette counties in southwestern Wisconsin. AGI risk was assessed for 10 viral, bacterial, and protozoan pathogens detected in private wells using quantitative microbial risk assessment. Exposure assessment was based on sampling 138 private wells in the study area, and risk was associated with fecal source by microbial source tracking (MST). Median Monte Carlo estimates indicated 4450 AGI cases/year in the study area associated with drinking water from contaminated private wells (95% confidence interval: 90–37,990). Most annual cases were associated with detection of human MST markers, including median estimates of 2550 associated with only human markers and 880 with human and livestock MST markers detected together. Note that 50 AGI cases/year were associated with detection of only livestock markers, and nearly 1000 cases were estimated to occur in the absence of detectable MST markers. This study characterizes AGI risk for households served by private wells in southwestern Wisconsin. It illustrates that human fecal sources can predominate risk associated with drinking water from private wells, even in a rural region characterized by substantial agricultural activity. MST can characterize the relationship between risk and fecal source; contributions of fecal sources to risk cannot be assumed from land use.