Different yeasts were isolated from spontaneous fermentation of Assyrtiko grape must in Santorini Island, Greece. Molecular typing revealed the presence of three Saccharomyces cerevisiae strains (S9, S13, S24) and one strain of the yeast species Nakazawaea ishiwadae (N.i). The four isolated strains were further tested in laboratory scale fermentations of Assyrtiko must in pure inoculation cultures and in sequential inoculation (72 hours) of each S. cerevisiae strain with the strain of N. ishiwadae. All fermentation trials were realised in duplicate.