Water microbial contamination is one of the major threats to human health. The study focus is on Infulene River Basin, a urban catchment with mainly informal settlements, with limited water supply and sanitation. In the catchment there are two wastewater treatment plants, one hospital and beer factory located on the banks of the main stream; water from this stream is used for urban agriculture and domestic uses by some dwellers. These factors present a significant health risk from water-borne diseases. At the moment there is limited knowledge about the level of microbial contamination of the different sources of water at the disposal of the communities. Thus, a preliminary study on fecal microbial contamination was conducted targeting the Infulene River and the drainage system from the nearby Maputo city draining into the system, with additional investigation on the drinking water provided by the city water supply company. The quantification of Total Coliforms (TC) and Escherichia coli (EC) was conducted at several sampling locations. Results were compared with official drinking water standards. Eighty two percent (82%) and 61% of Infulene river water and drainage water samples were positive for TC (105 to 109 NPN/100 mL) and EC (105 to 107 NPN/100 mL), respectively. For drinking water samples, 63% and 23% were positive for TC (up to 6000 NPN/100 mL) and EC (up to 1000 NPN/100 mL), respectively. Higher microbial contamination was found in neighborhoods with the poorest sanitation and shallow groundwater, i.e., Chamanculo, Xipamanine, Mafalala, Aeroporto and Maxaquene, a situation that was more expressive during the rainy season. Overall, the study confirmed the high vulnerability to microbial contamination of all sources investigated due to poor sanitation and lack of drainage infrastructure. The risks to human health might be even higher considering that contaminated water is used for gardening of vegetable watering and domestic use.