This study determined the comparative in vitro potency of the new investigative quinolone gemifloxacin (SB-265805) using low-passaged clinical isolates and type strains of mycoplasma commonly found in the human respiratory and urogenital tracts. Organisms studied were Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma fermentans, Mycoplasma genitalium, Mycoplasma penetrans and Ureaplasma urealyticum, obtained from different geographical regions. Comparator drugs were levofloxacin, trovafloxacin, grepafloxacin, azithromycin, clarithromycin, tetracycline and clindamycin. MICs were determined using a microbroth dilution method. The overall range of MICs of gemifloxacin was ≤ 0.008-0.125 mg/L for different Mycoplasma spp. and ≤ 0.008-0.5 mg/L for Ureaplasma spp. Depending on the species tested, gemifloxacin showed variable results when compared with the macrolides. However, gemifloxacin was as potent as, or more potent than, tetracycline, clindamycin and the other quinolones investigated.