Bacillus subtilis is commonly used as a biocontrol bacterium owing to its strong antifungal activity, broad-spectrum inhibition, and general safety. In this study, the inhibitory effects of volatile organic compounds (VOCs) produced by B. subtilis CF-3 on Colletotrichum gloeosporioides, a major destructive phytopathogen of litchi anthracnose, were analyzed using proteomics and transcriptomics. Differentially expressed genes (DEGs) and proteins (DEPs) indicated that the inhibition of C. gloeosporioides by B. subtilis CF-3 VOCs downregulated the expression of genes related to cell membrane fluidity, cell wall integrity, energy metabolism, and production of cell wall-degrading enzymes. Particularly, those important DEGs and DEPs related to the ergosterol biosynthetic and biosynthesis of unsaturated fatty acids are most significantly influenced. 2,4-di-tert-butylphenol, a characteristic component of B. subtilis CF-3 VOCs, also showed a similar effect on C. gloeosporioides. Our results provide a theoretical basis for the potential application of B. subtilis CF-3 in the postharvest protection of fruits and vegetables.