UV-vis, fluorescence, circular dichroism (CD) and 1H NMR spectroscopic techniques have been employed to explore the mode of binding of Mebendazole (MBZ) drug with calf thymus DNA (CT-DNA). UV-vis and fluorescence spectral studies suggested a complex formation between the drug and nucleic acid. The fluorescence of MBZ was found to enhance upon binding with CT-DNA through a ground state complex formation with Kb in the order of 104 M-1. The thermodynamic aspects indicated that the complex formation is a spontaneous process and an entropy-driven one. ΔH0 > 0 and ΔS0 > 0 revealed that hydrophobic interaction plays a dominant role in the stabilization of the complex. Competitive dye displacement assays with ethidium bromide (EB) and Hoechst 33258 dyes and viscosity measurements pointed out that MBZ binds with CT-DNA via intercalation mode, which is confirmed by CD and 1H NMR spectral studies as well as denaturation studies. Molecular docking analysis could not match well with the experimental results. However, molecular simulation studies and the resultant free energy surface (FES) analysis clearly showed that the benzimidazole ring of MBZ intercalated between the base pairs of the nucleic acid, which is in excellent agreement with the results of the various biophysical experiments.