Two billion people still consume drinking water contaminated with faeces. To improve this situation, it has been recognised by UNICEF and the WHO that a more rapid approach to detecting faecally contaminated drinking water is necessary. We have previously demonstrated that fluorescence spectroscopy is a significant real-time indicator of the presence/absence and number of faecal indicator bacteria in drinking waters in low-income countries of the tropics. We have also established its potential as an online indicator of faecal contamination of public water supplies in the UK. Outstanding questions remain, however, over the source of the fluorescence and its uniqueness to faecal-indicator bacteria. To address these, we sampled potable groundwater supplies in Kenya, Malawi, Senegal and Uganda across an urbanisation transect from rural Malawi through to the city of Dakar (Senegal) where pollution sources and pressures vary considerably. We report on whether the fluorescence signal in these sources is intracellular or extracellular and, in Senegal and Uganda, the ability of fluorescence spectroscopy to predict total bacteria cells and faecal-indicator bacteria.