Blue mold caused by Penicillium expansum is one of the most significant postharvest diseases of apples. Some microorganisms associated with the surface of ripen- ing apples possess the ability to inhibit the growth of P. expansum. However, the existing literature about their colonization in the stages before ripening is not explored in depth. This study aims to characterize the antagonistic capacity of bacterial populations from five fruit development stages of ‘Royal Gala’ apples. The re- sults have shown that the density of the bacterial popu- lations decreases throughout the ripening stages of fruit (from 1.0 × 105 to 1.1 × 101 cfu/cm2). A total of 25 bacte- rial morphotypes (corresponding to five genera identi- fied by 16S RNA) were differentiated in which Bacillus stood out as a predominant genus. In the in vitro an- tagonism tests, 10 Bacillus strains (40%) inhibited the mycelial growth of P. expansum from 30.1% to 60.1%, while in fruit bioassays, the same strains reduced the fruit rot ranging from 12% to 66%. Moreover, the bacterial strains with antagonistic activity increased in the ripening fruit stage. B. subtilis subsp. spiziennii M24 obtained the highest antagonistic activity (66.9% of rot reduction). The matrix-assisted laser desorption ioniza- tion-time of flight mass spectrometry analysis revealed that bacteria with antagonistic activity produce anti- fungal lipopeptides from iturin and fengycin families.