The antimicrobial effect of a benzoxazinorifamycin, KRM-1648, either alone or in combination with ofloxacin, was evaluated in vitro against two type strains and six clinical isolates of Mycobacterium ulcerans. Growth of M. ulcerans was measured by plate counts and the BACTEC radiometric method. The minimal inhibitory concentration as well as minimal bactericidal concentration of KRM-1648 against M. ulcerans was between 0.012 and 0.025 mg/l, while corresponding values for rifampicin and rifabutin were in the range of 0.1-0.8 mg/l and 0.1-0.4 mg/l respectively. When combined with ofloxacin, KRM-1648 exhibited strong synergistic activity while only additive effects were observed with the combination of rifampicin (or rifabutin) and ofloxacin. These results suggest that KRM-1648 has a great potential in the treatment of M. ulcerans infection.