Decreasing the ethanol content in wine is a current challenge, mainly due to the global climate change and to the consumer preference for wines from grapes with increased maturity. In this study, a central composite design (CCD) and response surface methodology (RSM) approach was used to investigate the potential application of Starmerella bacillaris (synonym Candida zemplinina) in combination with Saccharomyces cerevisiae, in mixed (co-inoculated and sequential) cultures, to understand better the mechanism of co-habitation and achieve the objective of reducing the ethanol in wines. Laboratory scale fermentations demonstrated a decrease up to 0.7 % (v/v) of ethanol and an increase of about 4.2 g/L of glycerol when S. cerevisiae was inoculated with a delay of 48 h with respect to the inoculation of S. bacillaris. Pilot-scale fermentations, carried out in winemaking conditions, confirmed the laboratory results. This study demonstrates that the combination of strains and inoculation protocol could help to reduce the ethanol content in wines.