Private water wells provide drinking water for an estimated 4.1 million households in Canada yet remain understudied in the context of microbial water quality or human health impacts. As there exists little systematic surveillance for enteric infections or outbreaks related to well water sources, consumers may be at risk of waterborne infectious diseases. A standard protocol in Ontario requires 200 mL of water, collected, and submitted by well owners, half of which is used to analyze for Escherichia coli and total coliforms (TCs). The aim of this study was to determine the efficacy of testing small water volumes and to survey for other contaminants in addition to bacterial indicators to inform pathogen prevalence and fecal source in drinking water wells. Samples were assessed for E. coli and TCs, by culture, and genetic markers of Bacteroides spp., Campylobacter spp., Salmonella spp., and Shiga toxin-producing E. coli, using qPCR. The source of fecal contamination varied by the geographic region and may be explained by septic tank density and underlying geology, among other factors. A small number of samples (1.9%) showed the evidence of contamination with enteric pathogens. Lastly, E. coli measured by qPCR, as opposed to culture, correlated more strongly to Bacteroides markers.