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A safe water supply is a necessity, but it remains one of the backlogs of services rendered in rural areas of developing countries. This leads to vulnerable communities using water from available sources that is unsafe as it is contaminated with faecal matter. Microbial source tracking (MST) methods are gold-standard techniques that detect the exact sources of faecal contamination. This study, therefore, tracked and identified the exact sources of faecal contamination from the catchment to the point of use in rural areas of Vhembe District Municipality. Collected water samples (n = 1048) were concentrated by membrane filtration for the enumeration and detection of E. coli, followed by DNA extraction. The extracted DNA was subjected to a quantitative polymerase chain reaction (qPCR) to track target host-specific Bacteroidales genetic markers from the water source to the point of use. Rivers and dams exhibited maximum E. coli counts of up to 90 CFU/100 mL during the wet season and up to 50 CFU/100 mL during the dry season. Due to the effective treatment of these water sources, no E. coli bacteria were detected in any of the sampled municipal drinking water treatment plants at the point of treatment, while this indicator bacterium was detected at the point of use (households), with a maximum of 4 CFU/100 mL recorded during both the wet and dry seasons. Overall, the most prevalent MST marker exhibited during the wet season was BacCan (dog-associated, 6.87%), followed by BacCow (cow-associated, 5.53%), while Pig-2-Bac (pig-associated, 2.48%) was the least prevalent. The most prevalent marker exhibited during the dry season was BacCan (5.34%), followed by BacCow, with Pig-2-Bac (1.72%) being the least prevalent. A positive correlation (r = 0.31, p = 0.001) was established between the presence of the MST markers and detected E. coli from water sources to the point of use. The knowledge of the faecal contamination attributes in both public and domestic domains will assist in developing prevention and control strategies. [ABSTRACT FROM AUTHOR]