Maintaining adequate acidity in wine is essential for microbial stability, colour preservation, and sensory quality—factors increasingly challenged by climate change, which promotes grape ripening conditions that reduce acidity and raise pH. Lachancea thermotolerans offers a promising biotechnological solution through its capacity to produce lactic acid during alcoholic fermentation. This study evaluated how different inoculation strategies—namely co-inoculation and sequential inoculation—using varying ratios of L. thermotolerans and Saccharomyces cerevisiae affected wine acidification and fermentative efficiency. Co-inoculations with higher proportions of L. thermotolerans led to significant increases in total acidity, achieving acidification levels comparable to those of sequential inoculation, without impairing fermentation kinetics. However, excessive acid production was associated with slower fermentations and elevated residual sugar levels. In addition to lactic acid, co-inoculation impacted the concentrations of other organic acids (e.g., malic and succinic acids), as well as glycerol content, contributing to the final acidity and mouthfeel of the wine. Notable shifts in volatile composition, including esters and higher alcohols, were also observed. These findings highlight the importance of optimising inoculation ratios in mixed fermentations to achieve a balance between acidification and fermentation performance, offering practical guidance for biological acidification under warming climate conditions.