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Resident fruit microflora has been the source of biocontrol agents for the control of postharvest decay of fruits and the active ingredient in commercialized biocontrol products. With the exception of grapes and apples, information on the resident microflora of other fruits is only fragmentary, but greater knowledge in this area can be very helpful in developing biocontrol strategies. We characterized the yeast microflora of nectarines (‘Croce del Sud’) from the early stages of fruit development until harvest. The fruit samples were collected from trees in an unmanaged orchard. The resident fruit microflora was separated from the occasionally deposited microorganisms by discarding initial fruit washings before the final wash, followed by sonication and plating on NYDA medium. The isolated yeasts were identified by BIOLOG and by sequencing the D1/D2 domain of a large subunit of the rRNA gene and, where available, the ITS sequence. BIOLOG identified 19 and the genetic analysis 23 species of yeasts. Although the identification by these two systems was not always the same, the predominant yeasts were Rhodotorula spp., Sporodiobolus spp., Cryptococcus spp., Pichia spp., Candida spp. and yeast-like Aureobasidium pullulans. Several of the taxa appear to represent new species. The preliminary biocontrol tests against brown rot of nectarine fruit caused by Monilinia fructicola indicates significant decay control potential of some of the identified yeast species, namely Cryptococcus magnus, Cryptococcus sp. nov., Sporidiobolus pararoseus, A. pullulans and Rhodotorula sp. nov.