Groundwater resources in Mahdia-Ksour Essef region, located in the central-eastern part of Tunisia, suffer from intensive exploitation and degradation of water quality. The assessment of groundwater vulnerability, the well-understanding of geochemical processes and the investigation of groundwater quality variations are of particular importance for water resources management in this semi-arid region. The results of this multi-disciplinary investigation show that groundwaters of Mahdia-Ksour Essef region are flowing from El Jem and Boumerdes regions in the South, towards the Mediterranean Sea and the sebkha of Moknine in the North. The groundwaters are classified as either Na-Cl or Ca-SO4. Stable oxygen and hydrogen isotope compositions (δ18O and δ2H) confirm a recharge by rapid water infiltration, evaporate surface water and water-rock interaction processes in the salinity increase. The rock-water interaction processes of the mineralization of groundwaters include the cation-exchange reactions and the dissolution of carbonates and evaporate. The high values of the water quality index (WQI) and of the total dissolved solids (TDS) (3.7–11 g/L) of these groundwaters indicate their unsuitability for drinking purpose. Moreover, the combination of the WQI, TDS and nitrate results prove the vulnerability of the studied groundwater to the anthropogenic pollution linked to agriculture and domestic activities and to the salt water contamination.