Enterobacter cloacae (MG001451) isolated from surface-sterilized parts of Ocimum sanctum showed potent antimicrobial activities against six bacterial human pathogens (Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Klebsiella pneumonia, Salmonella enterica, Pseudomonas aeruginosa), one bacterial phytopathogen (Xanthomonas oryzae) and two fungal phytopathogens (Pythium debaryanum and Rhizoctonia solani). The antibacterial activities were studied through Disc diffusion, Bacteriocin Like Inhibition Studies BLIS and Agar cup plate method and confirmed through Thin Layer Chromatography (TLC)-Bioautography. The Minimum Inhibitory Concentration (MIC) value of the extract of E. cloacae against the bacterial test organisms ranged from 0.39–12.5 mg/ml. Lower MIC and Minimum Microbicidal Concentration (MMC) values were found against Bacillus subtilis followed by Staphylococcus aureus. The isolate E. cloacae also inhibited the growth of Rhizoctonia solani and Pythium debaryanum, while studied through, dual culture and well plate assay method. Higher inhibition % was reported against Rhizoctonia solani(73.75 ± 2.5%) than Pythium debaryanum (60.24 ± 2.08%). Secondary metabolites like alkaloids, phenols, polychlorinated biphenyls, aromatic hydrocarbons, esters, etc. were identified in the extract through Gas Chromatography coupled Mass Spectrometry (GC-MS), which could be responsible for antimicrobial activities as observed during the investigation. © 2021 SAAB