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Resource-limited communities throughout the developing world face significant environmental health problems related to the myriad of coliform sources within those communities. This study comprehensively investigated contamination sources and the biological and chemical mechanisms sustaining them in two adjacent communities in rural Limpopo, South Africa. An 8-month study was conducted of household ( n = 14) and source water quality, measurements of biofilm layers on the inside of household water storage containers and water transfer devices, and also hand-based coliforms and hand-washing effectiveness. A 7-day water container incubation experiment was also performed to determine the biological and chemical changes that occur in a household water storage container independent of human interference. Results indicate that household drinking water frequently becomes contaminated after collection but before consumption (197 versus 1,046 colony-forming units/100 mL; n = 266; p < 0.001). The most important contamination sources include biofilm layers on the inside of storage containers (1.85 ± 1.59 colony-forming units/cm 2 ; n = 44), hands (5,097 ± 2,125 colony-forming units/hand; n = 48), and coliform regrowth resulting from high assimilable organic carbon (AOC) levels during storage. A maximum specific growth rate, μ max , of 0.072 ± 0.003 h -1 was determined for total coliform bacteria on AOC, and a high correlation between AOC concentrations and the growth potential of total coliform bacteria was observed. These results support the implementation of point-of-use water treatment and other interventions aimed at maintaining the safe water chain and preventing biological regrowth.