Abstract: Gray mold caused by Botrytis cinerea poses a serious threat to postharvest grape quality. To elucidate the antifungal activity of volatile organic compounds (VOCs) and their monomeric constituents from antagonistic actinomycete A89 against B. cinerea, this study identified strain A89 through morphological, physiological, biochemical, and molecular biological methods. Headspace solid-phase microextraction coupled with gas chromatography-tandem mass spectrometry (HS-SPME-GC-MS/MS) was employed to analyze its VOCs, followed by validation and identification of key antifungal components using pure standards. The inhibitory effects of A89-derived VOCs on gray mold in postharvest 'Red Globe' grapes were assessed using a sealed container system. Among four tested actinomycetes, A89 exhibited the highest inhibition rate (100%) against B. cinerea. Strain A89 was identified as Streptomyces microflavus. A total of 77 VOCs were detected during its 14-day growth, with major components including methyl 12-methyltridecanoate (18.59%), methyl palmitate (9.7%), methyl pentadecanoate (5.45%), heptadecane (4.64%), and 2,2,4-trimethyl-1,3-pentanediol diisobutyrate (4.57%). In vitro, assays revealed that nonanal and 2-ethylhexanol showed the strongest antifungal activity, with a minimum inhibitory concentration (MIC) of 7.81 μL/L against B. cinerea. In vivo, experiments demonstrated that after 10 days of fumigation, the 125 μL/L acetophenone treatment group completely suppressed gray mold incidence, followed by A89 VOCs and 500 μL/L 2,5-dimethylpyrazine, which reduced lesion diameters by 74.11% and 76.70%, respectively (P<0.05). These findings indicate that VOCs from S. microflavus A89 and its monomeric compound acetophenone exhibit significant potential for controlling postharvest gray mold in grapes.