The aim of this research is to identify the mineralization origin and the understanding of water flow between neighboring aquifer systems in the arid region: the Jeffara of Medenine (South Tunisia). This aquifer system is characterized by the presence of a faults network in stairs leading to hydrogeological relays and the configuration from unconfined to confined aquifer from west to east. The methodology used is the geological and hydrogeological survey as well as periodic sampling campaigns and interpretation of geochemical and isotopic groundwater data in the study area. Results demonstrated the hydraulic continuity between aquifer levels confirmed by the potentiometric map and the mineralization increase from upstream to downstream. The present study demonstrated that the origins of mineralization are sulphate salts dissolution (gypsum, anhydrite, epsomite, burkeite, thenardite, and mirabilite) and chloride (halite and magnesium chloride) associated with the crust. Results confirm communication between Zeuss-Koutine (ZK) and Sahel El Ababsa Triassic sandstone (TSE) aquifers, between ZK and Plio-Quaternary (PQ) aquifers, between PQ and Miocene (M) aquifers and the rainwater direct recharge of the three aquifers of the Jeffara groundwater system (TSE, ZK and PQ). These results can help decision makers to manage and protect the groundwater resources in South-Eastern Tunisia.