Climate change has caused sugar concentrations in grapes to rise, consequently leading to higher alcohol levels in wine. One of the strategies to mitigate ethanol production is the use of non-Saccharomyces yeasts. This study evaluated five separate sequential inoculation treatments, each involving a different non-Saccharomyces strain (M. guilliermondii P40D002, M. pulcherrima P01A016 or D11, W. anomalus O37, or L. thermotolerans O9), followed by a commercial Saccharomyces cerevisiae strain in chaptalized Chardonnay must. The final alcohol concentrations of the treatments were similar to those in fermentation using only S. cerevisiae. However, wines inoculated with M. pulcherrima P01A016 exhibited an alcohol reduction of 0.6–0.7% (v/v) compared to those inoculated with M. guillermondii, L. thermotolerans, or W. anomalus. Although the two M. pulcherrima strains had similar ethanol concentrations, their organic acid profiles, especially acetic and succinic acids, demonstrated significant variation, illustrating the importance of strain selection. Overall, diminished growth, YAN consumption, and the limitations of non-Saccharomyces strains in alcohol reduction indicate that high-sugar environments may pose challenges to their performance in the early stages of fermentation. The findings explain yeast performance under osmotic stress and highlight the importance of selecting non-Saccharomyces strains for alcohol reduction and the modulation of wine composition.