Surveys of the diatom assemblages from Aïn Bou Rkhiss and Aïn Kibrit, two springs that flow out into the Merguellil Wadi, and Aïn Ben Ali, a well located a few kilometers to the east, in the same geological formations (Kairouan district, Central Tunisia), were conducted in 2014 and 2016. The aim of this study was to characterize the species diversity of these water points, establish their ecological status and test the water quality using diatom indices standardized in France [Biological Diatom Index (BDI) and Specific Polluosensitivity Index (SPI)]. Several physical-chemical parameters were measured in 2016. Aïn Bou Rkhiss and Aïn Kibrit are in close proximity to one another but differ in terms of their bio-geochemical characteristics and uses (drinking water for domestic use and sulfur-rich water). We discuss the population’s growing anxiety with respect to their water supply and explore the traditional knowledge concerning the region’s groundwater circulation. The BDI index, reveals a lower quality than that assumed by local users. This may be due to a lack of knowledge about some other local water supplies of good quality that would have served as reference, and inadequacy of these methods designed for European freshwaters without consideration of more southern diatoms. A new variety of Luticola is described.