The checklist of the ascidian fauna (Tunicata: Ascidiacea) of Greece was compiled within the framework of the Greek Taxon Information System (GTIS), an application of the LifeWatchGreece Research Infrastructure (ESFRI) aiming to produce a complete checklist of species recorded from Greece. This checklist was constructed by updating an existing one with the inclusion of recently published records. All the reported species from Greek waters were taxonomically revised and cross-checked with the Ascidiacea World Database. The updated checklist of the class Ascidiacea of Greece comprises 75 species, classified in 33 genera, 12 families, and 3 orders. In total, 8 species have been added to the previous species list (4 Aplousobranchia, 2 Phlebobranchia, and 2 Stolidobranchia). Aplousobranchia was the most speciose order, followed by Stolidobranchia. Most species belonged to the families Didemnidae, Polyclinidae, Pyuridae, Ascidiidae, and Styelidae; these 4 families comprise 76% of the Greek ascidian species richness. The present effort revealed the limited taxonomic research effort devoted to the ascidian fauna of Greece, which is attributed to the lack of experts and low sampling effort. Therefore, major knowledge gaps on the ascidian diversity of Greece occur and further research in this field is needed.