Sulfites are considered the main additives in winemaking for their antimicrobial, antioxidant and anti-oxidasic activities. The current concern about the potential negative effects of sulfur dioxide (SO 2) on consumer health has focused the interest on replacing or reducing SO 2 use. Our work aims to develop a strategy based on the use of selected starter culture, able to perform wine fermentation without SO 2 addition. Four selected Saccharomyces cerevisiae indigenous strains were tested as mixed starter cultures in laboratory scale fermentations. The starter culture, characterized by a similar percentage of dominance of both strains composing the mixed starter and able to produce a wine characterized by the best combination of chemical and aromatic characteristics, was chosen. This mixed culture was tested as a starter at pilot scale with and without SO 2 addition, by using a higher inoculum level in the vinification without SO 2. The selected starter confirmed higher dominance ability in vinification without SO 2 addition than in SO 2-added fermentation, demonstrating that sulfite addition is not a guarantee to reach an absolute dominance of starter culture on indigenous microflora. The proposed biotechnological tool allowed to produce good quality wines possessing also “functional properties”, as NO-SO 2 added wines were characterized by high polyphenol content and antioxidant activity.