Monilinia fructigena is one of the most important fungal pathogens causing brown rot on apple and is heavily affecting fruit production. The main objective of this study was to screen for potential bacterial isolates with higher antagonistic activity against M. fructigena. Our study focused on the identification of potential bacterial isolates capable of reducing both the mycelial growth of M. fructigena and the disease severity using in vitro and in planta trials, respectively. To achieve this goal, thirteen bacteria, isolated from natural soil, were evaluated for their abilities to produce lytic enzymes (amylase, cellulase and protease), hydrocyanic acid (HCN) and lipopeptides (bacillomycin, fengycin, iturin and surfactin). Further, results from the dual culture method, volatile and bacterial free-cell filtrate bioassays indicated that tested isolates showed a fungicidal activity against the mycelial growth of M. fructigena. Thus, out of the 13 isolates tested, 12 exhibited significant mycelial inhibition (more than 70%) against M. fructigena, while remaining the last isolate displayed only a partial inhibition (up to 43%). Further, 12 of the bacteria isolates displayed an amylase production, 10 showed cellulase production, 11 revealed protease production, while only 2 displayed HCN production. In addition, most bacterial isolates were found to have genes encoding for different lipopeptides: bacillomycin (10), fengycin (3), iturin (11) and surfactin (1). Interestingly, two bacterial isolates, Bacillus amyloliquefaciens B10W10 and Pseudomonas sp. B11W11 were found to be the most effective and displayed the lowest disease severity in planta trial. These two bacteria reduced the brown rot incidence compared to the synthetic fungicide in a semi-commercial large-scale trial. Therefore, our findings suggest that these two later bacterial isolates provide apple protection against M. fructigena via direct and indirect mechanisms. These isolates may be used, therefore, as potential biological control agents (BCAs) in preventive treatment to control brown rot disease on apple fruits.