A group of four amphoras found in the anchialine cave of Vodeni Rat in the Croatian island of Sveti Klement, on a rocky ledge at 24 m water depth, indicates that a freshwater source was exploited at the bottom of this karstic pit by islanders and/or passing-by sailors from the late Roman Republican Period to the Early Medieval Period. In other words, prior to the 4th–7th century CE, Vodeni Rat was not an anchialine cave but a Pleistocene karstic pit with a freshwater pool at the bottom. Seawater started to infiltrate this cavity via newly opened fissures in the surrounding limestone after the 4th–7th century CE, as the archeometric age of the youngest intact amphora found down in the pit suggests. At present, the 29-m-deep water body is stratified, with a saline water layer at the bottom, a freshwater layer at the surface, and a transitional brackish water layer in the middle. Our study of the physical and biological characteristics of the cave water column revealed diversified and highly partitioned populations of stygobitic crustaceans including ostracods, copepods, amphipods, and thermosbaenaceans. Some species known to be exclusively endemic of highly saline habitats were collected from the bottom saline layer, whereas others known to be of freshwater origin were found in the upper water layer. This suggests that the freshwater dwellers were already present in this cave prior to the early medieval event that caused seepage of seawater into this karstic pit.