To study the effectiveness of moxifloxacin (MOX) against multidrug-resistant (MDR) Mycobacterium tuberculosis (MTB), in vitro drug susceptibility tests on MOX and ofloxacin (OFX) using the MGIT system against 132 nonduplicate MDR MTB isolates (108 OFX-sensitive and 24 OFX-resistant) were performed. Eleven OFX-resistant non-MDR MTB isolates were also included. All strains that were susceptible to OFX were shown to be also susceptible to MOX. For OFX-resistant isolates, regardless of their MDR status, a 4- to 8-fold decrease in MIC was observed for MOX when compared to OFX. On the basis of similarities in mode of action and pharmacokinetic data between MOX and OFX, a breakpoint MIC of 2 mg/L was suggested for MOX in the MGIT system. All but two OFX-resistant isolates possessed MIC of MOX of 2 mg/L or lower and were considered to be susceptible to MOX; however, all OFX-resistant strains studied had mutations involved in resistance to quinolones. DNA sequence analysis for the gyrase A mutation, which was an indicator for OFX resistance, correlated with MOX susceptibility changes. Significant decreases in MOX susceptibility amongst MDR-MTB strains were observed that correlated with OFX susceptibility. The Asp94Gly mutation appeared to be associated with a higher level of MICs to both OFX and MOX. These findings on the stepwise decline of in vitro susceptibility to MOX suggest that clinical usage of MOX should be accompanied by careful monitoring of susceptibility to this important anti-MDR MTB drug.