Abstract: Currently the criteria used for selecting optimal new antibacterial drug candidates include inhibitors of nucleic acid synthesis, fatty acid biosynthesis and folic acid pathway. The aim of the present study was to study the interaction of the isolated anti-bacterial compound, 2, 5-Di-tert-butyl-1, 4-benzoquinone (DTBBQ) from Streptomyces sp. VITVSK1 with 4 selected antibacterial drug target enzymes by in silico molecular docking approach. The compound DTBBQ showed minimum binding energy of -3.91 kcal mol-1 with Topoisomerase II, with Topoisomerase IV-3.24 kcal mol-1 with, Enoyl ACP reductase -4.51 kcal mol-1 and 5.82 kcal mol-1 with Dihydrofolate reductase protein. The compound DTBBQ interacted with several amino acid residues, of which lysine was found to be common among all the target enzymes. The results of our study suggest that DTBBQ could be used for antibacterial activity by targeting bacterial proteins of drug resistant strains.