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Hydro-(major and trace elements: Cd, F and Sr), isotope (¹⁸O, ²H, ³H and ¹³C) geochemistry and radiogenic carbon (¹⁴C) of dissolved inorganic carbon (DIC) were used to investigate the sources of groundwater contamination and the hydrodynamic functioning of the multilayer aquifer system in the mining Gafsa basin (Southwestern Tunisia). The groundwater of the study area is subject to intense exploitation to accommodate all the water demands of this arid area. The Gafsa basin contains a multi-layered aquifer with four principal levels: Upper Zebbag (Cenomanian–Turonian), Abiod (Campanien–Maastrichian), Beglia (Miocene) and Segui (Plio–Quaternary) Formations. The hydrogeology of this system is largely affected by tectonics (Gafsa–Tebessa, Sehib, Negrine–Tozeur, Tabeddit and Metlaoui faults…). The groundwater of these aquifers undergoes a significant decline in water level (≈0.5 m y⁻¹ …