5-Nitroisatin is used for the treatment of fungal and bacterial strains exploring antimicrobial activity. Structural and vibrational spectroscopic studies were carried out by using FT-IR, FT Raman, UV and NMR spectral analysis together with DFT method using GAUSSIAN'09 software. Assignments of vibrational spectra have been carried out with the aid of NCA. NBO analysis, Charge Analysis, HOMO LUMO, ESP, aromaticity and thermodynamic properties have been analyzed. Thermal stability has been analyzed by TG/DTA method. 5NI has been screened for its antimicrobial activity and found to exhibit antifungal and antibacterial effects. Docking simulation has been performed. (C) 2015 Elsevier B.V. All rights reserved.