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Abstract Background Tomato yield and quality are influenced by multiple factors, including conditions during cultivation, post-harvest handling and storage. Botrytis cinerea, the fungal pathogen causing gray mold disease, poses a significant threat to tomato production. This disease poses a serious challenge to tomato cultivation on a global scale, leading to significant crop losses and compromising food supply security. The purpose of this study was to assess the efficacy of three different bacterial bioagents, namely Pseudomonas fluorescens, Bacillus amyloliquefaciens and Pseudomonas chlororaphis, against B. cinerea. Results From the rhizosphere of a tomato field, 22 bacterial isolates were collected. Among them, three isolates, designated as 1, 8 and 11, displayed notable antagonistic effects against Botrytis cinerea during in vitro testing and were selected for further analysis. The bacteria were identified as Pseudomonas fluorescens, Bacillus amyloliquefaciens and P. chlororaphis through 16S rRNA gene sequencing. The experiments demonstrated that these three isolates significantly inhibited the growth of the pathogenic fungus and promoted plant growth, with B. amyloliquefaciens emerging as the most effective inhibitor, displaying the highest antifungal activity. In vitro and greenhouse experiment suggest that the bacterial isolates hold considerable promise as natural antagonists for managing gray mold disease in tomatoes. The study further recommends using all three isolates for controlling the gray mold, with B. amyloliquefaciens showing the most significant potential, evidenced by its consistently lower disease severity scores, achieving the highest % efficacy (85.75) compared to control. Conclusions The study emphasizes the significant potential of P. fluorescens, B. amyloliquefaciens and P. chlororaphis as effective bioagents to control tomato gray mold disease, B. cinerea. By directly targeting the fungal pathogen, these isolates provide an effective strategy for mitigating the adverse impacts of gray mold, contributing to higher crop yield and quality. Further research needs on open field trials and commercialization strategies for large-scale application of these bacterial bioagents.