This study explores tomato agri-food residues as sources of bacteria with bioprotective potential to enhance product shelf-life and safety. A total of 245 bacterial strains were isolated, comprising predominantly Pseudomonas (52%) and Bacillus (44%) spp., with lactic acid bacteria (LAB) present at lower levels (4%). The antimicrobial activity of these isolates was assessed against pathogenic and spoilage bacteria and phytopathogenic molds. Notably, the Bacillus isolate TRB1-7 exhibited moderate activity against L. monocytogenes (inhibition halo diameter: 10.64 mm), while Pseudomonas and LAB isolates showed limited or no inhibition. Antifungal assays highlighted significant antifungal potential for Bacillus isolates. Results showed that 16% and 15% of the 245 isolates inhibited F. oxysporum and C. acutatum growth, respectively. Nine of these isolates underwent acid-adaptation and were evaluated against the selected molds using Potato Dextrose Agar (PDA) at pH 4.0 to simulate tomato conditions. Only isolate BRZ3-2, identified as B. aerius, was adapted to acidic conditions and inhibited F. oxysporum by 25%. Experiments on tomato-based agar at the same pH showed no inhibition by Bacillus isolates. These results suggest that tomato microbiota harbors acid-tolerant Bacillus strains with potential for post-harvest bio-preservation. Further studies on strains TRB1-7 and BRZ3-2 are required to develop effective bioprotective applications.