Groundwater storage in the Euro-Mediterranean karst region has been detected to be decreasing across approximately 60–80% of its area, yet only a few studies have attempted to quantify this phenomenon. This study conducts a spatio-temporal characterization of karst aquifer dynamics on a continental scale using Gravity Recovery and Climate Experiment (GRACE) measurements. The groundwater storage anomaly (GWSA) and subsurface water storage anomaly (SWSA) were computed from GRACE data, with GWSA representing water storage in the saturated zone and SWSA encompassing storage in both unsaturated and saturated zones. We systematically analyzed and compared GWSA and SWSA to explore groundwater recharge and storage dynamics. The analysis was complemented by VarKarst model simulations, which represent potential groundwater recharge in karst terrain, and validated using spring discharge observations from selected karst catchments. Trend analysis of both GWSA and the simulated potential karst groundwater recharge for the period 2002–2019 revealed the steepest decline in groundwater recharge and storage in the Alpine region's polar climate zone. In temperate climate zones, a strong correlation was found between SWSA and the simulated potential karst groundwater recharge when recharge values were timely shifted. The results highlight the significant role of soil and epikarst in influencing karst aquifer recharge, particularly in temperate climates, and demonstrate the spatial suitability of GRACE satellite data for large-scale karst system characterization. The observed declining trends in GWSA underscore the importance of sustainable groundwater management in the Euro-Mediterranean karst region. Spatial variations in GWSA reflect the impacts of climate, water use, and characteristic karst properties on groundwater storage dynamics. This study emphasizes the need to consider the effects of the epikarst when relating GRACE data to recharge processes in karst systems. Overall, the integrated approach combining GRACE measurements, karst groundwater recharge modeling, climate data, spring discharge observations, and water use information provides a robust methodology for characterizing continental-scale karst hydrogeology.