Corn stalk rot, caused by Fusarium graminearum, severely threatens corn production, while chemical fungicides lead to resistance and environmental risks. Thus, exploring environmentally friendly biocontrol agents is crucial. Though Bacillus polyketones have antifungal activity, their active components, corn stalk rot biocontrol potential and rhizosphere impacts remain unclear. This study isolated polyketones from Bacillus velezensis DJ1, assessed their antifungal activity and mechanism against F. graminearum, verified greenhouse efficacy, analyzed rhizosphere microbiota, and identified active components via HPLC-HDMS Q-TOF. The results showed that DJ1 polyketones showed dose-dependent inhibition by disrupting F. graminearum’s mycelium and membrane, with 44.33% greenhouse control efficacy. They did not disturb rhizosphere balance, only increasing Bacteroidota and DYGX01, and the active components were difficidin and bacillaene. These findings confirm DJ1 polyketones as promising eco-friendly biocontrol agents, providing a new strategy for sustainable corn stalk rot management.