Groundwater is valued as a source of potable water, although it is vulnerable to environmental pollution. The aim of this study was to track enteric pathogen contamination from on-site sanitation (OSS) facilities to 70 household boreholes used by four villages of the Vhembe District Municipality. Two objectives were pursued: to measure the lateral distance between the borehole and the sanitation facilities in household yards, and to track the enteric pathogens. The borehole abstraction point and OSS system distance were determined using a steel measuring tape. Real-time PCR was used to track Shigella flexneri, Salmonella typhimurium, Campylobacter jejuni, and enterotoxigenic Escherichia coli (ETEC) from Wastewater (WW) from domestic septic tank and sludge from pit latrines to boreholes. Escherichia coli was used as an indicator of faecal contamination. Results showed that 25% of households kept a distance of >= 50 m between the OSS facilities and the boreholes. In total, 87.5% of household boreholes in the rainy season and 72.5% in the dry season were contaminated with E. coli and pathogenic bacteria: Shigella flexneri, Salmonella typhimurium, and ETEC. The concentrations of the pathogens ranged from 2.03 to 2.12 LogEGC/100 mL. A very weak (r = -0.093) to moderate (r = -0.541) association was found between pathogens in groundwater and on-site sanitation systems (WW from septic tank and sludge from pit latrine). This suggests that the pathogens were not present in the sanitation compartment when they were found in the groundwater and vice versa. Moreover, a very weak (r = 0.007) to moderate (r = 0.525) association was found between the detected contaminants in groundwater and the lateral distance between the OSS facilities and the boreholes. The pathogens detected in all samples showed consistent concentrations, suggesting potential contamination from OSS systems' waste, possibly in groundwater, indicating potential contamination. The siting of OSS facilities at the yards in this study appeared to have a slight influence on the contaminants detected in groundwater. This study calls for an education program to be implemented by the Water and Sanitation Services Authorities to prevent contamination of groundwater and the risk of waterborne diseases.
