This study focuses on identifying, selecting, and multiplying indigenous yeast species of technological interest in the Stefan Voda Protected Geographical Indication grape region. Based on microbiological studies, the multiplication of non-Saccharomyces yeast species: Hanseniaspora, Kloeckera, and Torulaspora, was performed under sterile conditions, with inoculum prepared from sterile, fresh grape must. To assess how the inoculum prepared from indigenous yeasts affects the wine’s physicochemical properties, aromatic profile, and sensory qualities, two grape varieties, Muscat and Traminer Rose (Vitis vinifera), were used. Standard and microbiological analyses of the wines produced by two established processes showed that the presence of non-Saccharomyces yeasts during the initial fermentation stage (days 1 to 3), followed by inoculation with S. cerevisiae, enhances wine complexity and increases glycerol and 2,3-butanediol levels. The results from yeast microbiome correlation and PCA analysis of the two fermentation methods clearly distinguished the wines produced with mixed-sequencing fermentation (sample II) from the control samples. This indicates that the process improves microbiological stability, develops a more complex aromatic profile, and aligns with tasters’ preferences while maintaining the wine’s authenticity linked to its specific geographical origin.
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