Abstract <p>The biological characteristics of promising <i>Bacillus subtilis</i> M-22 and <i>B. subtilis</i> I-5 strains— antagonists of phytopathogenic fungi and bacteria—were characterized in laboratory model and pot experiments. First- and second-generation clones of the original <i>B. subtilis</i> I-5 isolate, differing in the level of antagonistic activity, were obtained. For the first time, the composition of culture filtrates of strain B. subtilis M-22 and first- and second-generation clones of the psychrotolerant strain <i>B. subtilis</i> I-5 was studied using MIC-filtration based on mass ranges of chromatograms obtained by high-resolution HPLC-MS using an IT-TOF mass spectrometer. This approach allowed for the identification of components unique to each strain and the precise determination of their molecular masses, which, in turn, enabled the proposal of the most probable molecular formulas. By correlating the component composition of each filtrate with its biological activity, compounds most likely responsible for specific biological activities were identified.</p>