The mycobacterial mel2 locus (mycobacterial enhanced infection locus, Rv1936-1941) is Mycobacterium marinum and M. tuberculosis specific, which can withstand reactive oxygen species (ROS) and reactive nitrogen species (RNS) induced stress. A library of over a million compounds was screened using in silico virtual ligand screening (VLS) to identify inhibitors against the modeled structure of MelF protein expressed by melF of mel2 locus so that M. marinum's ability to withstand ROS/RNS stress could be reduced. The top ranked 1000 compounds were further screened to identify 178 compounds to maximize the scaffold diversity by manually evaluating the interaction of each compound with the target site. M. marinum melF was cloned, expressed and purified as maltose binding protein (MBP)-tagged recombinant protein in Escherichia coli. After establishing the flavin dependent oxidoreductase activity of MelF (~ 84 kDa), the inhibitors were screened for the inhibition of enzyme activity of whole cell lysate (WCL) and the purified MelF. Amongst these, 16 compounds could significantly inhibit the enzyme activity of purified MelF. For the six best inhibitory compounds, the minimal inhibitory concentration (MIC) was determined to be 3.4-19.4 M and 13.5-38.8 M for M. marinum and M. tuberculosis, respectively. Similarly, the minimal bactericidal concentration (MBC) was determined to be 6.8-38.8 M and 27-38.8 M against M. marinum and M. tuberculosis, respectively. One compound each in combination with isoniazid (INH) also showed synergistic inhibitory effect against M. marinum and M. tuberculosis with no cytotoxicity in HeLa cells. Interestingly, these inhibitors did not display any non-specific protein-structure destabilizing effect. Such inhibitors targeting the anti-ROS/RNS machinery may facilitate the efficient killing of replicating and nonreplicating mycobacteria inside the host cells.