Copyright © 2015, American Society for Microbiology. All Rights Reserved. In this study, susceptibilities were determined for AZD0914, a spiropyrimidinetrione DNA gyrase inhibitor, azithromycin, doxycycline, and levofloxacin against Mycoplasma and Ureaplasma species. The activity of AZD0914 was comparable to that of levofloxacin and doxycycline against Mycoplasma genitalium and Mycoplasma pneumoniae. The AZD0914 MIC90 against Mycoplasma hominis was 8-fold greater than that for levofloxacin. The AZD0914 MIC90 against Ureaplasma species was 4-fold less than that for azithromycin and 8-fold less than that for levofloxacin and doxycycline.