Mycoplasma genitalium is a sexually transmitted reproductive tract pathogen of men and women. M. genitalium infections are increasingly difficult to treat due to poor efficacy of doxycycline and acquired resistance to azithromycin and moxifloxacin. A recent clinical trial suggested that metronidazole may improve cure rates for women with pelvic inflammatory disease and reduced the detection of M. genitalium when included with standard doxycycline plus ceftriaxone treatment. As data regarding susceptibility of mycoplasmas to nitroimidazoles are lacking in the scientific literature, we determined the susceptibility of 10 M. genitalium strains to metronidazole, secnidazole, and tinidazole. MICs ranged from 1.6 to 12.5 μg/mL for metronidazole, 3.1 to 12.5 μg/mL for secnidazole, and 0.8 to 6.3 μg/mL for tinidazole. None of these agents was synergistic with doxycycline in checkerboard broth microdilution assays. Tinidazole was superior to metronidazole and secnidazole in terms of MIC and time-kill kinetics and was bactericidal (>99.9% killing) at concentrations below reported serum concentrations. Mutations associated with nitroimidazole resistance were identified by whole-genome sequencing of spontaneous resistant mutants, suggesting a mechanism for reductive activation of the nitroimidazole prodrug by a predicted NAD(P)H-dependent flavin mononucleotide (FMN) oxidoreductase. The presence of oxygen did not affect MICs of wild-type M. genitalium, but a nitroimidazole-resistant mutant was defective for growth under anaerobic conditions, suggesting that resistant mutants may have a fitness disadvantage in anaerobic genital sites. Clinical studies are needed to determine if nitroimidazoles, especially tinidazole, are effective for eradicating M. genitalium infections in men and women.