Karst systems constitute valuable freshwater resources in Crete island, a region of Greece that is threatened by water scarcity. The present work refers to evaluation of the available groundwater potential in a karst terrain and contributes to providing adequate quantities of fresh water to the central southern Crete (Rethimno prefecture). The available groundwater potential was estimated by combining conventional hydrogeological approaches and an analysis of hydrographs of Kourtaliotis spring that drains the system. The research procedure contributed to the three-dimensional understanding of the karst system and provided reasonable estimates regarding the groundwater reserves in the area. The geological (permanent) storage in the karstified system was estimated to 415 × 106 m3 while the dynamic reserves were calculated equal to 43 × 106 m3. Based on the research results, it is considered possible to pump annually an amount of 21–29 × 106 m3 over the quantities of water which naturally outflow from Kourtaliotis spring, in order to satisfy the water demands in the region. The study provides a valuable guidance on predicting the groundwater reserves in aquifers with similar hydrogeological regime.