Using an ultra performance liquid chromatography system, the presence of seven aromatic carboxylic acids, known as antimicrobial substances, has been established in root exudates of barley colonized by the phytopathogenic fungus Fusarium culmorum and the antagonistic bacterium Pseudomonas fluorescens. It was established that barley produced fewer antimicrobial components in the exudates in response to the colonization of P. fluorescens, than after colonization by F. culmorum. All aromatic carboxylic acids inhibited the growth of F. culmorum, but only two of them inhibited the growth of P. fluorescens, and only in the highest concentration. The obtained data testify to the ability of the plant to create favorable conditions for the development of beneficial rhizobacteria and unfavorable for the growth of the phytopathogenic fungus via composition of root exudates. In the future, it is possible to select varieties of grain crops with a specific composition of antimicrobial components in root exudates.