Macrolide or fluoroquinolone-resistant Mycoplasma genitalium is spreading worldwide. We aimed to determine the influence of single nucleotide polymorphisms (SNPs) in the quinolone resistance determining regions (QRDR) of parC and gyrA in cultured M. genitalium strains. In addition, we examined the prevalence of macrolide- and fluoroquinolone resistance mediating mutations in specimens collected from Japanese male patients with urethritis in two time-periods between 2005–2009 and 2010–2017, respectively, by sequencing the QRDR of parC and gyrA and domain V of the 23S rRNA gene. The minimum inhibitory concentrations (MIC) of moxifloxacin, sitafloxacin, ciprofloxacin, levofloxacin, doxycycline, minocycline, azithromycin and clarithromycin were determined in 23 M. genitalium strains. Three cultured strains had elevated MICs for moxifloxacin at 16, 4 and 2 mg/L and had SNPs with the amino-acid change Ser83→Ile in ParC ( p<0.001) and 3 kinds of SNPs with amino-acid changes Asp99→Asn, Gly93→Cys and Met95→Ile in GyrA, respectively. Among a total of 148 M. genitalium positive urine specimens, the prevalence of A2058G and A2059G SNPs in the 23S rRNA gene and any SNPs in ParC increased from 4.8% and 22.6% in 2005–2009 to 42.2% and 53.1% in 2010–2017, respectively. If M. genitalium is considered multi-drug resistant in clinical specimens carrying SNPs in the 23S rRNA gene and Ser83→Ile in ParC, the prevalence of multi-drug resistance is 12.5% in 2010–2017 in Japan. In conclusion, the SNP resulting in Ser83→Ile in ParC is closely related to moxifloxacin resistance even though other factors may also affect treatment outcomes by moxifloxacin. The prevalence of circulating multi-drug resistant M. genitalium strains with macrolide- and fluoroquinolone-resistance is dramatically increasing in Japan.