In this study, medicinal plant (Solanum surattense)-associated bacteria were isolated and their extracellular secondary metabolites were extracted. Dual-plate application of crude secondary metabolites proved that SSL2I and SSL5 had a good inhibitory activity against Ralstonia solanacearum. These biocontrol bacteria were identified as Bacillus subtilis and Bacillus velezensis by 16S rRNA gene sequencing analysis. The crude extracts of secondary metabolites were identified based on high-resolution liquid chromatography/mass spectrometry (HR-LCMS) analysis. On the basis of HR-LCMS analysis, we selected the compounds such as glucosamine and indole acetaldehyde for in silico analysis and inhibition of pathogenic gene of phcA from R. solanacearum. The specificity of identified pathogenic gene of R. solanacearum and its cytoplasmic localization were identified by BLASTP and PSORTB bioinformatics tools. The protein-protein interaction between the identified secondary metabolites and pathogenic gene revealed that the compound had antagonistic potential against pathogenic gene of phcA. Furthermore, the synthetic forms of the above metabolites showed that both the compounds had the ability to inhibit R. solanacearum under in vitro condition. On the basis of in silico and in vitro analyses, it was concluded that medicinal plant-associated Bacillus spp. could be used as a biocontrol agent in managing wilt disease caused by R. solanacearum.