Background: Verticillium dahliae, the causal agent of Verticillium wilt, is notoriously invasive in many crops and has been involved in numerous epidemics worldwide. Bacillus species, as representatives of biocontrol bacteria, produce a variety of lipopeptides (LPs), which are useful as biofungicides to many pathogenic fungi, including Verticillium dahliae. This study will explore the mechanism of resistance of V. dahliae to Bacillus and biocontrol bacteria.Results: By using in vitro confrontation bioassays, we found that under the stress induced by Bacillus, the spore vitality of V. dahliae with larger colonies was higher, and more abundant microsclerotia were formed. Then, according to the RNA-Seq analysis, the target of rapamycin (TOR) and mitophagy pathways were enriched among the significantly upregulated 542 genes observed in two co-culture groups with different colony sizes. In addition, in the group of V. dahliae with large colonies, the pathways related to cell wall synthesis, microsclerotia formation and the clearance of reactive oxygen species were regulated, and the expression of genes was up-regulated.Conclusion: This study found that the larger colonies of V. dahliae were more resistant to the antagonistic actions of Bacillus and the likelihood of the formation of homeostasis. Therefore, the prevention of Verticillium wilt by Bacillus is more effective than the treatment of an active fungal infection. These transcriptomic insights provide direction for the use of fungicides in the prevention and treatment of diseases such as Verticillium wilt.