Acetobacter syzygii CCTCC M 2022983 was isolated and characterized from Tibetan kefir grains, which is utilized as a functional food with diverse bioactive properties. After 6 days of fermentation by A. syzygii, Acetobacter fermented extract (AFE) showed significantly higher antioxidant, anti-tyrosinase, and anti-melanin effects compared to the unfermented yeast extract (UFY). Western blotting confirmed that AFE reduced melanogenesis-related proteins (MITF, TYR, TRP-1, TRP-2). LC-MS/MS analysis identified 4-hydroxybenzoic acid as abundant in AFE, contributing to its antioxidant capacity. Succinic acid and citric acid emerged as the major compound and a type of mixed inhibitor against mushroom tyrosinase, with IC50 values of 2.943 mM and 1.615 mM, respectively. Fluorescence spectra analysis revealed that these acids caused conformational changes in tyrosinase. Moreover, succinic acid and citric acid prevented L-DOPA from auto-oxidation with IC50 values of 0.355 mM and 0.261 mM, respectively. Molecular docking analysis suggested that these acids interacted with the association of the H and L subunits of tyrosinase, thereby reducing its stability. In B16-F10 cells, succinic and citric acids significantly reduced melanin production in a dose-dependent manner. Thus, succinic acid and citric acid revealed promising potential for applications in the food and medicine industries as melanogenesis inhibitors due to their safety.