Ascidian spicules are reported for the first time from the upper Eocene (Paleogene) biosiliceous marls and mudstones of the Blanche Point Formation in the St. Vincent Basin, southeastern Australia. The spicules that belong to the family Polycitoridae are identified as a Recent species Cystodytes cf. dellechiajei, and Cystodytes sp. and spicules of the family Didemnidae are indentified as representing genera Lissoclinum, Didemnum, and Polysyncraton. Five other different morphological spicule types which can be classified only at the family level also belong to the Didemnidae. This study demonstrates that ascidians had a very wide distribution by the late Eocene and that ascidian fauna was already of a modern character.