Groundwater in the Mooi River catchment is prone to mining, agricultural, municipal and septic tank pollution. In this study physico-chemical and microbiological parameters were determined using appropriate methods. Bacterial isolates were identified by 16S rRNA sequencing (heterotrophic plate count (HPC) bacteria and amoeba-resistant bacteria (ARB)) and multiplex polymerase chain reaction (Escherichia coli). Antibiotic resistance tests were also performed. Physico-chemical parameters were generally within target water quality ranges for drinking water. HPC bacteria ranged between 105 and 107 colony-forming units (cfu)/ml. E. coli were enumerated from Trimpark, School and Cemetery. The Blaauwbank borehole was negative for faecal streptococci. Pseudomonas spp. were most abundant in the bulk water. Opportunistic pathogens isolated included Pseudomonas aeruginosa, Acinetobacter, Aeromonas, Alcaligenes, Flavobacterium, Bacillus cereus and Mycobacterium spp. Varying patterns of antibiotic resistance were observed. Most HPC bacterial isolates were resistant to cephalothin and/or amoxicillin and a few were resistant to erythromycin and streptomycin. Pseudomonas spp. was also the most abundant ARB. Other ARBs included Alcaligenes faecalis, Ochrobactrum sp. and Achromobacter sp. ARBs were resistant to streptomycin, chloramphenicol, cephalothin, and/or amoxicillin compared to HPCs. The presence of E. coli and ARB in these groundwater sources indicates potential human health risks. These risks should be further investigated and quantified, and groundwater should be treated before use.