Groundwater management is crucial, particularly in arid and semi-arid regions, where it is influenced by the climate and human activities. The Gabès region in southeastern Tunisia is known for its significant groundwater resources, which are mainly associated with the Continental Intercalary (CI) and Djeffara aquifers as well as interlayers from the Ceno-Turonian and Miocene-Pliocene aquifer systems. The Cretaceous and Quaternary aquifer systems are the most exploited in this region. This study aimed to assess the hydrochemistry of these aquifer systems and contribute to understanding the recharge mechanisms. To achieve this, 44 groundwater samples were collected and analyzed for major ions and the isotopes oxygen-18 and deuterium. The results indicate that the total dissolved solids concentration ranges from 300 to 7200 mg/L, with temperatures varying from normal (19 °C) to extremely high (> 70 °C). Cluster analysis revealed two main groups (A and B) with distinct chemical and isotopic characteristics. The isotopic ratios of oxygen (δ 18 O) and hydrogen (δ 2 H) were used to identify the recharge mechanism. The findings suggest relatively depleted 18 O and 2 H isotopic ratios, indicating a mixing of groundwater from the Djeffara aquifer with deep thermal groundwater from the CI. However, groundwater depth and local geological structures also influence the mixing pattern. The results highlight the need for detailed structural characterization in the future to identify the spatial distribution and dominant properties of fractures. This information is crucial for understanding their contribution to regional groundwater recharge. Integrating hydrogeochemical data and the isotopic groundwater composition will enhance our understanding of the geochemical processes governing groundwater hydrochemistry and recharge in the Gabès region as well as in similar hydrogeological and structural settings.