Background: Considering the potentials of gatifloxacin and moxifloxacin in the treatment of tuberculosis, the present study was aimed to define resistance to both these drugs. Methods: Fifty Mycobacterium tuberculosis isolates, consisting of 30 ofloxacin-susceptible and 20 ofloxacin-resistant strains, were tested for their susceptibility to gatifloxacin and moxifloxacin using different susceptibility testing methods, namely the absolute concentration method on Lowenstein-Jensen medium (LJ), the proportion susceptibility testing method (PST) on LJ and 7H11 agar media, and the BACTEC radiometric method. Result: The minimal inhibitory concentration (MIC) of gatifloxacin and moxifloxacin was 1 µg/ml by the absolute concentration method on LJ. In the PST method on LJ and 7H11, using a criterion of ≧1% growth as resistant, there was 100% agreement with the absolute concentration method at a concentration of 0.5 µg/ml for gatifloxacin, and 96% agreement with the BACTEC method at a concentration of 0.25 µg/ml. For moxifloxacin, results by the PST method showed 96% agreement with the absolute concentration method on LJ at a concentration of 1 µg/ml and 92% agreement at a concentration of 0.5 µg/ml for both the absolute concentration method on 7H11 and the BACTEC method. Conclusions: The MICs of gatifloxacin and moxifloxacin were much lower than the MICs of other quinolones like ofloxacin and ciprofloxacin. Additionally, these two drugs have shown a low mean MIC and low concentration as a definition of resistance, which might help in treating the patients with low levels of quinolone resistance.