The morphology of fossil remains of Vestalenula (Darwinuloidea, Ostracoda) from Crete (Greece), Serbia and Italy were analysed using morphometric valve variability analysis. Three new species were distinguished and described, Vestalenula angulata n. sp., Vestalenula longissima n. sp. and Vestalenula pliocenica n. sp., and represent the first record of this genus in the Neogene of Italy and Serbia. Vestalenula angulata n. sp. was recorded from the early Messinian of Tuscany, Italy (Baccinello and Velona Basins), Vestalenula longissima n. sp. was collected in the Upper Pliocene (Gelasian) sediment core drilled in the Sabina area (Latium, Italy), and Vestalenula pliocenica n. sp. was collected from the Early Tortonian of Vrysses (Rethymnon Basin, Crete), from the lower Messinian deposits of the Velona Basin (Tuscany, Italy), in the Late Pliocene of the Paludinian Beds (Middle Danube Plain) and Mazgoš deposits (Serbia), and in the Upper Pliocene deposits of the Sabina area (Latium, Italy). Despite the freshwater character of most living species of Vestalenula, the fossil species here analysed were found associated with halotolerant forms, showing they could also withstand oligohaline conditions.