The effects of Metschnikowia pulcherrima and high glucose osmolality on S. cerevisiae pyruvate dehydrogenase pathway (PDH) by-pass were examined by varying the starting sugar concentration of synthetic grape juice and the inoculation ratio of S. cerevisiae to M. pulcherrima. The findings revealed that M. pulcherrima and osmolarity impacted S. cerevisiae’s PDH by-pass. The inoculation concentration of M. pulcherrima significantly affected pyruvate decarboxylase (PDC) activity and acs2 expression when the initial sugar concentration was 200 g L⁻¹ and 290 g L⁻¹. The osmolarity caused by the initial sugar (380 g L⁻¹) significantly influenced the enzymatic activity of S. cerevisiae, which decreased PDC and acetaldehyde dehydrogenase (ALD) activities while increasing Acetyl-CoA synthetase (ACS) activity. The reduction in acetic acid in the wine was caused by M. pulcherrima altering the initial sugar concentration faced by S. cerevisiae, which in turn affected enzymatic activity. The alteration of enzyme activity and accumulation of primary metabolites revealed why mixed fermentation could reduce the acetic acid content in wine by altering the enzymatic activity and affecting the expression of several key genes. The M. pulcherrima inoculation levels had no significant effect on the acetic acid and glycerol concentration in the same fermentation medium.