Levonadifloxacin (WCK 771) was evaluated against 68 type strains and clinical isolates of Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma pneumoniae, and Ureaplasma spp. in comparison with moxifloxacin, levofloxacin, tetracycline, and azithromycin or clindamycin. Levonadifloxacin MICs were ≤0.5 μg/ml for M. genitalium MIC90s were 1 μg/ml for M. hominis, 0.125 μg/ml for M. pneumoniae, and 2 μg/ml for Ureaplasma spp. Levonadifloxacin merits further study for treating infections caused by these organisms.