The majority of the root microbiota formation derives from soil-dwelling microorganisms. The limited extent of thorough investigation leads to a dearth of knowledge concerning the intricate mechanisms of microbe-microbe interaction implicated in the establishment of root microbiota. Therefore, the taxonomic signatures in bacterial inhibition profiles were determined by in vitro testing of 39,204 binary interbacterial interactions. However, findings from genetic and metabolomic studies elucidated that co-functioning of the antimicrobial 2,4-d iacetylphloroglucinol (DAPG) and the iron chelator pyoverdine as exometabolites has significantly contributed to the potent inhibitory activities of the highly antagonistic Pseudomonas brassicacearum R401. Microbiota restoration with a core of Arabidopsis thaliana root commensals showed that these exometabolites possess a root niche-specific function in establishing root competence and inducing anticipated changes in root surroundings. Both biosynthetic operons are abundant in roots in natural habitats, indicating that these exometabolites co-functioning is an adaptive feature that helps Pseudomonad dominate the root microbiota.