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The sub-arid southwest of Madagascar is one of the world’s biodiversity hotspots combined with exceptional poverty and high risk of further temperature increase that will aggravate the living and health conditions of the people. As bases for future water management, we measured the physicochemical and microbiological quality of water sources across different forms of land use in the protected Tsimanampetsotsa National Park, and the agricultural and pastoral regions of the Mahafaly plateau on limestone and the coastal plain on sand during the dry and wet season of 2012-2013. We investigated spatial and seasonal variation of water characteristics and their relationships with bacterial contamination. Portable meters were used for the physicochemical measures. The compact dry method was used for microbial analyses. The pH was neutral to slightly alkaline and within the permissible limits of WHO and Malagasy standards. Electric conductivity (EC) and total dissolved solids (TDS) were very high and above the permissible limits in the coastal plain, moderately high in the park and low on the plateau. The concentrations of nitrogen components were high in the rainy season, with the highest concentrations in wells. Phosphate concentration was high throughout the study area. Total coliforms, Escherichia coli, Salmonella spp. and Vibrio spp. were present throughout the study area year-round, representing a serious health hazard. Their concentrations were not correlated with any physicochemical characteristics in any systematic fashion that would allow to use the physicochemical characteristics as proxy for microbial contamination. Poor sanitary conditions are the principal causes of the water contamination that could be reduced substantially by simple behavioural changes of the local human population. The finding that water temperature in wells of the plateau and to a lesser extent of the coastal plain increases during the hot wet season indicates a substantial contribution of surface rather than subterranean water to the water available for human and livestock consumption. This limits the options for future increase of water consumption by people, livestock and agriculture in the region.