© 2017 American Society for Microbiology. All Rights Reserved. Gepotidacin, a novel first-in-class triazaacenaphthylene topoisomerase II inhibitor, was tested against 85 type strains and clinical isolates of Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma parvum, and Ureaplasma urealyticum in comparison to levofloxacin, moxifloxacin, azithromycin or clindamycin, and tetracycline. Gepotidacin MIC90s (g/ml) were 0.125 (M. pneumoniae), 0.032 (M. genitalium), 2 (M. hominis), and 8 (Ureaplasma species). Gepotidacin activity was not affected by resistance to fluoroquinolones, tetracyclines, or macrolides in the strains tested. Gepotidacin merits further study for treating infections caused by these organisms.