Over one million households rely on private water supplies (e.g. well, spring, cistern) in the Commonwealth of Virginia, USA. The present study tested 538 private wells and springs in 20 Virginia counties for total coliforms (TCs) and Escherichia coli along with a suite of chemical contaminants. A logistic regression analysis was used to investigate potential correlations between TC contamination and chemical parameters (e.g. NO3−, turbidity), as well as homeowner-provided survey data describing system characteristics and perceived water quality. Of the 538 samples collected, 41% (n = 221) were positive for TCs and 10% (n = 53) for E. coli. Chemical parameters were not statistically predictive of microbial contamination. Well depth, water treatment, and farm location proximate to the water supply were factors in a regression model that predicted presence/absence of TCs with 74% accuracy. Microbial and chemical source tracking techniques (Bacteroides gene Bac32F and HF183 detection via polymerase chain reaction and optical brightener detection via fluorometry) identified four samples as likely contaminated with human wastewater.