Nemonoxacin, a newly developed non-fluorinated quinolone (NFQ), selectively inhibits bacterial DNA topoisomerase activity. However, its activities against
Mycoplasmas
have rarely been studied to date. Herein, the activities of nemonoxacin were evaluated against clinical isolates of 50
Mycoplasma pneumoniae
, 20
Mycoplasma hominis
, and 77
Ureaplasma
spp., and they were compared to fluoroquinolones, tetracyclines, and macrolides. Nemonoxacin MICs (μg/ml) ranged from 0.03 to 0.25 for
M. pneumoniae
, 0.25 to 8 for
M. hominis
, and 0.06 to >16 for
Ureaplasma
spp., and all of the ranges are similar to those of fluoroquinolones. The activity of nemonoxacin against
Mycoplasmas
was not affected by resistance to macrolides in the strains tested, but it seems to have the same resistant mechanism as fluoroquinolones. In addition, minimum bactericidal concentrations (MBC) of nemonoxacin to
M. pneumoniae
were within two dilutions of the MIC values, indicating a bactericidal effect on
M. pneumoniae
. Nemonoxacin merits further study for treating infections caused by these organisms.