The in vitro susceptibilities to garenoxacin (BMS-284756), an investigational des-fluoroquinolone, and eight other agents were determined for 63 Mycoplasma pneumoniae, 45 Mycoplasma hominis, 15 Mycoplasma fermentans, and 68 Ureaplasma sp. isolates. Garenoxacin was the most active quinolone, inhibiting all isolates at ≤1 μg/ml. The garenoxacin MIC at which 90% of isolates are inhibited (MIC90s; ≤0.008 μg/ml) was at least 4-fold less than those of moxifloxacin and clindamycin, 8-fold less than that of sparfloxacin, and 64-fold less than those of levofloxacin and ciprofloxacin for M. pneumoniae. For M. hominis, the garenoxacin MIC90 (≤0.008 μg/ml) was 4-fold less than those of clindamycin and moxifloxacin, 8-fold less than that of sparfloxacin, and 64-fold less than those of levofloxacin and ciprofloxacin. All 15 M. fermentans isolates were inhibited by garenoxacin at concentrations ≤0.008 μg/ml, making it the most active drug tested against this organism. For Ureaplasma spp., the garenoxacin MIC90 (0.25 μg/ml) was equivalent to those of moxifloxacin and doxycycline, 4-fold less than those of levofloxacin and sparfloxacin, 8-fold less than that of azithromycin, and 32-fold less than that of ciprofloxacin. Garenoxacin and the other fluoroquinolones tested were demonstrated to have bactericidal activities against M. pneumoniae and M. hominis by measurement of minimal bactericidal activities and by time-kill studies. Further study of garenoxacin is required, as it has great potential for use in the treatment of infections due to mycoplasmas and ureaplasmas.