Rice sheath blight, caused by Rhizoctonia solani, is a major constraint to sustainable rice production, yet
mechanistic understanding of effective biological control remains limited. In this study, a mycolytic bacterial
strain, Bacillus cereus strain KT-9, was evaluated for its antifungal activity and underlying mechanisms. Strain KT-
9 completely inhibited R. solani growth in dual culture assays (100% inhibition) and significantly suppressed
fungal growth in culture filtrates, with 93.4% inhibition at 70% (v/v), demonstrating broad-spectrum antifungal
activity. Its culture filtrates showed activity comparable to prochloraz and higher than azoxystrobin, while
retaining full activity after autoclaving and suppressing sclerotial germination, indicating the presence of heatstable
bioactive metabolites. LC–QTOF–MS analysis revealed diverse putatively identified metabolites, including
cyclic peptides, peptide-derived metabolites, aromatic acid derivatives, and maculosin, which may contribute to
the observed antifungal activity. Exposure of R. solani to strain KT-9 culture filtrates induced oxidative stress, as
indicated by increased reactive oxygen species levels, elevated antioxidant enzyme activities, and disruption of
glutathione redox balance. In greenhouse experiments, strain KT-9 treatments enhanced rice growth, increasing
plant height by 57% (33.8 cm vs. 21.5 cm in control) and reducing disease severity by 55% compared with
pathogen-inoculated controls. Overall, these findings suggest that strain KT-9 suppresses rice sheath blight
through multiple interacting mechanisms, including production of heat-stable antifungal metabolites, induction
of oxidative stress in the pathogen, and direct mycolytic activity. This study highlights the potential of B. cereus
strain KT-9 as a sustainable biocontrol agent and provides insight into its antifungal mechanisms.