Our objectives were to explore the prevalence of Chlamydia trachomatis, Neisseria gonorrhoeae, and Mycoplasma genitalium in Japanese female commercial sex workers (CSWs), in comparison with pregnant women as controls. A high-risk group of 174 female CSWs and 90 asymptomatic pregnant women were enrolled in this study. Detection of C. trachomatis, N. gonorrhoeae, and M. genitalium on the endocervix of the women was performed mainly by using polymerase chain reaction (PCR)-based assays. The prevalence rates of C. trachomatis, N. gonorrhoeae, and M. genitalium were 19.0%, 32.8%, and 12.6%, respectively, in the CSWs, compared with 5.6%, 0%, and 1.1% respectively, in the pregnant women. These results suggest a high prevalence of C. trachomatis, N. gonorrhoeae, and M. genitalium in Japanese CSWs. We conclude that continued close monitoring of the prevalence of C. trachomatis, N. gonorrhoeae, and M. genitalium infection in CSWs is important for preventing the dissemination of these microorganisms, and that further investigation of M. genitalium as a sexually transmitted pathogen in women is needed.