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There a lot of studies including the use of non-Saccharomyces yeasts in the process of wine fermentation. The attention is focused on the first steps of fermentation. However, the processes and changes that the non-Saccharomyces yeast populations may have suffered during the different stages of grape berry ripening, caused by several environmental factors, including antifungal treatments, have not been considered in depth. In our study, we have monitored the population dynamics of non-Saccharomyces yeasts during the ripening process, both with biochemical identification systems (API 20C AUX and API ID 32C), molecular techniques (RFLP-PCR) and enzymatic analyses. Some unusual non-Saccharomyces yeasts have been identified (Metschnikowia pulcherrima, Aureobasidium pullulans, Cryptococcus sp. and Rhodotorula mucilaginosa). These yeasts could be affected by antifungal treatments used in wineries, and this fact could explain the novelty involved in their isolation and identification. These yeasts can be a novel source for novel biotechnological uses to be explored in future work.