Nitro substituted benzimidazole derivatives were synthesized using o-phenylenediamine and different aromatic acid. The reaction is carried out in ethanol as solvent at room temperature by using mechanical stirrer. Nitration of the synthesized benzimidazole derivative was carried out using mixed acid as a nitrating agent. The synthesized product characterized by using FT-IR, and 1H NMR. The synthesized benzimidazole derivative were studied for antimicrobial activity using gram -ve and gram +ve micro-organism. Maximum zone of inhibition is 18mm for B. cereus gram +ve microorganism and 17 mm for E. coli gram -ve microorganism in comparison with streptomycin as control drug. In silico studies were adopted for synthetic derivatives by Chem bioDraw, and online software tool and the synthesized compounds XY-1, XY-2 and XY-3 showed good binding affinity than streptomycin. The results suffice in vitro studies.