We evaluated the in vitro antimicrobial activity of NM394 and ofloxacin (OFLX) against representative pathogenic mycobacteria by the agar dilution method, using 7H11 agar medium. NM394 showed appreciable antimicrobial activity against Mycobacterium tuberculosis (MIC90=0.78μg/ml), M. kansasii (MIC90=6.25μg/ml), M. marinum (MIC90=3.13μg/ml) and M. fortuitum (MIC90=3.13μg/ml), whereas the agent was not active against M. scrofulaceum (MIC90=>100μg/ml), M. avium (MIC90=50μg/ml), M. intra-cellulare (MIC90=<100μg/ml), M. chelonae subsp. abscessus (MIC90=<100μg/ml) and M. chelonae subsp. chelonae (MIC90=25μg/ml). The in vitro antimicrobial activity of the agent against M. fortuitum was a little more active than that of OFLX, whereas the activity of NM394 against the other mycobacteria was slightly inferior to that of OFLX. The antimycobacterial activity of NM394 against M. tuberculosis H37Rv (MIC: NM394=0.78μg/ml, OFLX=0.78μg/ml) phagocytosed in murine peritoneal macrophages was lessactive than that of OFLX, when the macrophages were cultured in RPMI-1640 medium containing 1μg/ml or 10μg/ml of these agents for up to 5 days.