Abstract Aims This study investigates the traits of three plant growth-promoting (PGP) and antagonistic bacteria, Pseudomonas protegens MP12, Bacillus sp. 3R4, and Bacillus sp. T22, to assess their potential application as biocontrol agents by using the ecofriendly and low-cost substrate Corn Steep Liquor (CSL) medium. Analyses of antagonism through volatile organic compounds (VOCs) production, biofilm formation, and growth performance were carried out. Methods and results Dual antagonism assay showed that all strains displayed significant antagonistic activity against Botrytis cinerea through VOCs. Gas chromatography demonstrated that strains in the CSL exhibited higher VOCs production than nutrient medium. Moreover, enhanced biofilm formation analysed by Calgary Biofilm Device, growth, and biomass were noted in CSL cultures. Pseudomonas protegens MP12, which showed higher cell concentration and biomass yield, was selected for freeze-drying treatments. Storage cell viability assays evidenced that it can be effectively preserved for nearly 7 months at 4°C. Conclusion The results here obtained showed that CLS medium enhanced VOCs production, biofilm formation, growth, and biomass of the antagonistic bacteria of the three strains. Eventually, the more effective strain P. protegens MP12 can be stored for nearly 7 months at 4°C.