FT-IR and FT-Raman spectra of 5-nitro-2-phenoxymethylbenzimidazole were recorded and analyzed theoretically and experimentally. The splitting of N-H stretching mode in the IR spectrum with a red shift from the calculated value indicates the weakening of the NH bond. The theoretical calculations give the phenyl ring breathing modes at 999 cm(-1) for mono substituted benzene ring and at 1040 cm(-1) for tri-substituted benzene ring. The theoretical NMR chemical shifts are in agreement with the experimental chemical shifts. The most reactive sites for electrophilic and nucleophilic attack are predicted from the MEP analysis. HOMO of pi nature is delocalized over the entire molecule whereas the LUMO is located over the complete molecule except mono-substituted phenyl ring and oxygen atom. Reactive sites of the title molecule have been located with the help of ALIE surfaces and Fukui functions. In order to determine locations prone to autoxidation and locations interesting for starting of degradation, bond dissociation energies have been calculated for all single acyclic bonds. For the determination of atoms with pronounced interactions with water we have calculated radial distribution functions obtained after molecular dynamics simulations. The calculated first hyperpolarizability of the title compound is 58.03 times that of standard nonlinear optical material urea. The substrate binding site interactions of the title compound with Topo II enzyme is reported by using molecular docking study. Biological activity studies show that the title compound can be leaded for developing new anticancer agents. (C) 2016 Elsevier B.V. All rights reserved.