The study aimed at assessing the microbiological quality and suitability of roof-harvested rainwater (RHRW) for crop irrigation and domestic use. In total, 80 rain water tanks (246 samples) across three rural villages (Ga-Molepane, Jericho and Luthngele) were visited. Culture based techniques were used to isolate bacterial microbes and identities were confirmed using matrix assisted laser desorption/ ionization time of flight (MALDITOF MS). Uncultured fungal populations were also identified using pyrosequencing. Salmonella spp. (3%), L. monocytogenes (22%), total coliforms (57.7%), Escherichia coli (E. coli) (30.5%), Enterococcus spp. (48.8%), Pseudomonas spp. (21.5%) were detected in RHRW samples after rainfall. Fungal sequences belonging to species known to cause fever, coughing and shortness of breath in humans (Cryptococcus spp.) were identified. This study indicates that RHRW quality can be affected by external factors such as faecal material and debris on the rooftops. The use of untreated RHRW could pose a potential health risk if used for irrigation of crops or domestic use, especially in the case of a relative high population of immunocompromised individuals. This study does not dispute the fact that RHRW is an alternative irrigation water source but it recommends treatment before use for domestic purposes or for watering crops.