Access to clean and potable water is a great challenge in most rural areas of South Africa’s Mpumalanga Province. The aim of this study was to conduct a preliminary investigation to determine whether the quality of the groundwater supply poses a possible threat to the health of these communities. Groundwater samples collected from 100 boreholes in rural areas of Mpumalanga were subjected to culture-based methods and molecular analysis targeting 16S RNA gene. The physical and chemical contents were also determined. The results of the study revealed that all the physico-chemical parameters were within the limits set by the national guidelines for domestic purposes, with the exception of magnesium, calcium, fluorides, nitrate, and turbidity. Seventy percent of the boreholes complied with the fluoride limit (0-1 mg/l), 98% with the nitrate (0-6 mg/l as N), 85% with the magnesium (0-30 mg/l as Mg), 68% with the calcium (0-32 mg/l as Ca, and 52% with turbidity (<1 NTU). The results also indicated that 78% and 81% of the boreholes did not comply with the limits set by the national guidelines in terms of fecal (0 cfu/100 ml) and total coliforms (0-5 cfu/100 ml), respectively. Of 100 boreholes, molecular study revealed the presence of Citrobacter freundii in 35%, Serratia marcescens in 19%, Bacillus cereus in 11%, Enterobacter cloacae in 9%, Salmonella enterica in 7%, and Pseudomonas maltophilia in 7%. Escherichia coli O157:H7, Shigella flexineri, Klebsiella oxytoca, and Cronobacter sakazakii were found in 1% of 100 boreholes. The results of this study were conclusive evidence that some groundwater supplies in rural areas of Mpumalanga pose a serious threat to the health of consumers.