The discoveryof new antimicrobial agents is extremely needed to overcome multidrug-resistant bacterial and tuberculosis infections. In the present study, eight novel substituted urea derivatives (10a-10h) containing disulfide bond were designed, synthesized and screened for their in vitro antimicrobial activities on standard strains of Gram-positive and Gram-negative bacteria as well as on Mycobacterium tuberculosis. According to the obtained results, antibacterial effects of the compounds were found to be considerably better than their antimycobacterial activities along with their weak cytotoxic effects. Molecular docking studies were performed to gain insights into the antibacterial activity mechanism of the synthesized compounds. The interactions and the orientation of compound 10a (1,1 '-((disulfanediylbis(methylene))bis(2,1-phenylene))bis(3-phenylurea)) were found to be highly similar to the original ligand within the binding pocket E. faecalis beta-ketoacyl acyl carrier protein synthase III (FabH). Finally, a theoretical study was established to predict the physicochemical properties of the compounds.