When two antibacterial agents act simultaneously upon a uniform microbial population, the result may be addition, synergism, antagonism or indifference.Interest in the use of combination of antibiotics was started many years ago, almost as soon as two antibiotics were available. This study is carried out to identify in vitro effective combination of Isoniazide (INH) and Rifapmicin (RF) with Isoxyl (ISO) against   MDR and susceptible strains of Mycobacterium tuberculosis and Mycobacteria Other Than Tuberculosis (MOTT) strains by checkerboard titration using Microplate Alamar Blue Assay( MABA) in terms of Minimum Inhibitory Concentration( MIC) values.Total 20 MDR strains , 10 susceptible strains of M. tuberculosis and Four MOTT strains were  included in the study.When INH & ISO and RF & ISO were used in combination against resistant and susceptible strains of M. tuberculosis and MOTT strains, enhancement of inhibitory activity of INH and RF was observed. Results are depicted by Fractional Inhibitory concentration Index (FIC).When FIC index is less than or equal to 0.5, combination is synergistic .When FIC index is more than or equal to 2.0, combination is antagonistic. When FIC index is equal to 1.0, combination is additive.  The MICs of INH dropped more than RF along with ISO against resistant and susceptible strains of M. tuberculosis and MOTT strains. There was an in vitro synergistic interaction between ISO & INH and ISO & RF to M.tuberculosis and MOTT strains.ISO and INH combination was more synergistic than ISO and RF combination.It appears that ISO had biological effect in the prevention of emergence of resistant strains, to standard drugs and as a corollary, must be efficacious as an antituberculosis drug in combination therapy.