[Background] Fungal diseases have gradually become a major factor restricting the crop production in China, and thus it is urgent to develop antifungal agents for disease prevention and control. [Objective] To investigate the mechanism of Bacillus subtilis J-15 secondary metabolites (SMs) in inhibiting fungal growth and development at the molecular level, so as to provide a theoretical basis for their application in fungal disease control. [Methods] The cell viability of Saccharomyces cerevisiae S288C treated with SMs was measured, and the cell necrosis was detected by flow cytometry. Transcriptome sequencing was performed to analyze the effects of SMs on the gene expression in S. cerevisiae, and real-time fluorescence quantitative PCR was employed to verify the results. [Results] SMs caused nuclear membrane lysis, DNA diffusion, and cell necrosis of S. cerevisiae S288C in a time-dependent manner. A total of 1 627 differentially expressed genes were screened out after the treatment with SMs for 12 h, including 851 up-regulated genes and 776 down-regulated genes. A total of 512 genes were differentially expressed after the treatment with SMs for 24 h, including 300 up-regulated genes and 212 down-regulated genes. The differentially expressed genes were involved in multiple pathways such as autophagy, sugar, lipid, and amino acid metabolism, cell wall synthesis, and cell cycle. [Conclusion] The findings provide an experimental basis for revealing the mechanism of SMs in inhibiting fungal growth and a theoretical basis for the application of SMs in the prevention and control of crop fungal diseases.