show Abstracthide AbstractAspergillus flavus is a notorious plant pathogen responsible for the contamination of agricultural crops, with peanut (Arachis hypogaea) being particularly susceptible to aflatoxin infection. This susceptibility underscores the urgent need for the development of more effective treatment methods. In this study, we isolated the strain Penicillium baileys W2 from the rhizosphere soils of healthy peanut seedlings. The fermentation extract of this strain demonstrated a concentration-dependent inhibition of pathogenic A. flavus growth, with a minimum inhibitory concentration (MIC) of 55% and a minimum fungicidal concentration (MFC) of 60%. Physiological data and transcriptome analysis revealed that the W2 fermentation supernatant inhibited A. flavus growth by disrupting membrane permeability. Metabolomics was utilized to identify the active ingredients in the fermentation solution, which were subsequently validated through plate tests. The results indicated that active compounds such as propylparaben, taxifolin, and phloretin exhibited significant antagonistic effects against A. flavus. Furthermore, we assessed the impact of the W2 cell-free fermentation broth on peanut growth promotion and the structure of the rhizosphere microbial community using soil metagenomic sequencing. The reduction of harmful microorganisms in the soil contributed to the maintenance of soil health, while the increased abundance of beneficial microorganisms enhanced peanut seedling growth by promoting the transformation of soil nutrient cycles. These findings suggest that the development and application of strain P. baileys W2 or its fermentation extract are aligned with the principles of sustainable agriculture and represent a promising biological control strategy.