Environmental stable and radioisotopes have been employed to identify the origin, the recharge mode and geochemical processes controlling groundwater quality. In southeastern Tunisia, where population is growing and water demand for domestic and irrigation activities is increasing, water balance assessment constitutes a serious concern about sustainability of water resources. Isotopic characterization using stable isotopes (δ2H, δ18O and δ13C) and radiocarbon (14C) of the Djeffara aquifer system lead to characterization of carbon origin in the system and identification of geochemical processes that control water chemistry. In addition, statistical methods were used to identify the distribution of the salinity. Multivariate statistical analysis (principal component analysis “PCA” and hierarchical cluster analysis “HCA”) revealed the main sources of groundwater mineralization. The main reactions responsible for the hydrochemical evolution in the Djeffara groundwater fall into three categories: (1) mixing process; (2) dissolution of salts; and (3) cation exchange process. Djeffara groundwater shows markedly low 14C activities, confirming that the aquifer has limited modern recharge. The modelling of isotopic evolution of the dissolved inorganic carbon (DIC) pool indicates the presence of additional source of carbon (δ13C ≈ −5‰) typical for mantle CO2 which can explain the variability of δ13C and 14C activities observed in DIC pool of groundwater samples.