To improve mulberry vinegar flavor, single-strain (SS) fermentation using Acetobacter pasteurianus HN 1.01 was combined with mixed-strain (MS) fermentation using Acetobacter pasteurianus HN 1.01 and Torulaspora delbrueckii NJSYGA 2021. The physicochemical properties, organic acid composition, and volatile flavor compounds of SS and MS fermented mulberry vinegar were systematically analyzed. The results showed that MS exhibited a higher total acid content and a more rapid reduction in residual sugars than SS. Compared with SS, MS exhibited significantly increased concentrations of L-malic (9.73%), L-lactic (11.80%), acetic (22.58%), citric (8.70%), and succinic (31.18%) acids. In total, 87 volatile flavor compounds were detected across samples, with MS exhibiting superior diversity and relative abundances, particularly of alcohols and esters. A total of 15 differential volatile flavor compounds were identified, including phenylethyl alcohol, isobutanol, ethyl caprate, and ethyl laurate. Co-occurrence network analysis revealed that correlations among volatile compounds were stronger in MS than in SS, especially among phenylethyl alcohol, 2-nonanol, isobutanol, and 3-furfuryl alcohol. Differential metabolites were enriched in the glyoxylate and dicarboxylate metabolism, propionate metabolism, pyruvate metabolism, glycolysis/gluconeogenesis, and phenylalanine metabolism pathways. Overall, MS fermentation significantly improved mulberry vinegar flavor, providing a basis for further optimization of mulberry vinegar production.