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The pied de cuve (PdC) technique involves using a portion of grape must to undergo spontaneous fermentation, which is then used to inoculate a larger volume of must. This allows for promoting autochthonous yeasts present in the must, which can respect the typicality of the resulting wine. However, the real impact of this practice on the yeast population has not been properly evaluated. In this study, we examined the effects of sulphur dioxide (SO 2), temperature, ethanol supplementation, and time on the dynamics and selection of yeasts during spontaneous fermentation to be used as PdC. The experimentation was conducted in a synthetic medium and sterile must using a multi-species yeast consortium and in un-inoculated natural grape must. Saccharomyces cerevisiae dominated both the PdC and fermentations inoculated with commercial wine yeast, displaying similar population growth regardless of the tested conditions. However, using 40 mg/L of SO 2 and 1% (v/v) ethanol during spontaneous fermentation of Muscat of Alexandria must allowed the non- Saccharomyces to be dominant during the first stages, regardless of the temperature tested. These findings suggest that it is possible to apply the studied parameters to modulate the yeast population during spontaneous fermentation while confirming the effectiveness of the PdC methodology in controlling alcoholic fermentation. • Stress and inoculation time were analysed in pied de cuve fermentations. • Temperature, SO 2, and ethanol have strong impact on the yeast species community. • Temperature has the highest impact on fermentation rate and length. • The selected factors favour the diversity of non- Saccharomyces first and Saccharomyces at the latter. [ABSTRACT FROM AUTHOR]