Groundwater constitutes the principal water supply source in the Jenin Governorate, facilitating various household, agricultural, and industrial uses. Groundwater is mostly sourced from many wells around the region, with the main sources being two significant aquifer systems: the Jenin Subseries aquifer, dating to the Eocene epoch, and the Mountain aquifer, which is part of the Turonian–Upper Cenomanian geological era. These aquifer systems are especially vulnerable to contamination from various anthropogenic and environmental influences. Significantly, pollution sources encompass untreated wastewater discharge, industrial effluents, agricultural runoff laden with fertilizers and pesticides, and ongoing soil degradation. The permeability of the top lithological layers influences the danger of contamination. Although groundwater pollution is prevalent, successful remediation poses significant technical and economic challenges. Consequently, the sustainable and strategic management of groundwater quality is critically important. This study utilized the Groundwater Vulnerability Matrix (GVM) method to assess the vulnerability of groundwater resources in the Jenin Governorate. The GVM approach comprises seven essential parameters: (1) aquifer type, (2) a depth to groundwater, (3) aquifer lithology, (4) proximity to recharge zones, (5) probable pollution sources inside recharge zones, (6) characteristics of the surface layer, and (7) total aquifer storage capacity. The assessment results reveal that the unconfined Eocene aquifer demonstrates significant susceptibility to contamination, chiefly attributable to its shallow water table and porous geological properties. Areas with shallow groundwater levels were designated as extremely vulnerable, while regions with deeper groundwater levels were classified as highly vulnerable. The unconfined segment of the Turonian–Upper Cenomanian aquifer exhibits moderate susceptibility, whilst the confined sections of same aquifer are regarded as having a medium level of vulnerability.