Botrytis cinereais a necrotrophic fungal pathogen with an extremely broad host range, causing significant economic losses in agricultural production. In this study, we discovered a culture filtrate of bacterial strain HK235, which was identified asChitinophaga flava, exhibiting high levels of antifungal activity againstB. cinerea. From the HK235 culture filtrate, we isolated a new antimicrobial peptide molecule designated as chitinocin based on activity-guided fractionation followed by characterization of the amino acid composition and spectroscopic analyses. The HK235 culture filtrate and chitinocin completely inhibited both conidial germination and mycelial growth ofB. cinereaat a concentration of 20% and 200 μg/mL, respectively. In addition to antibiosis againstB. cinerea, the active compound chitinocin had a broad antifungal and antibacterial activityin vitro.When tomato plants were treated with the culture filtrate and chitinocin, the treatment strongly reduced the development of gray mold disease in a concentration-dependent manner compared to the untreated control. Here, considering the potent antifungal propertyin vitroandin vivo, we present the biocontrol potential ofC. flavaHK235 for the first time.