Mycoplasma hominis and Ureaplasma urealyticum are important causative agents of vaginitis, cervicitis, postpartum sepsis, reproductive infections and infertility in both males and females.According to the uncertain prevalence of U. urealyticum and M. hominis in Iranian infertile females, the present study was carried out to determine the prevalence of U. urealyticum and M. hominis in high vaginal swab samples of fertile and infertile females.A total of 350 high vaginal swab specimens were taken from fertile and infertile females. Samples were cultured and those that were positive for bacteria were subjected to the polymerase chain reaction (PCR) for further confirmation.Of the 350 collected samples, eleven were positive for M. hominis (3.14%), fifteen were positive for U. urealyticum (4.28%) and five were positive for both of them (1.42%). Prevalence of U. urealyticum and M. hominis in the high vaginal parts of infertile females was higher than fertile females (P < 0.05). The results of traditional method were also confirmed, using the PCR amplification of urease gene of U. urealyticum and 16SrRNA gene of the M. hominis. Ureaplasma urealyticum and M. hominis had a higher prevalence in the high vaginal samples collected during the summer season.Considerable prevalence of M. hominis and U. urealyticum in the high vaginal swab samples of infertile females compared to the low prevalence in fertile females may suggest that these two pathogens can be cause infertility. Application of the PCR method is recommended for rapid and sensitive detection of M. hominis and U. urealyticum in high vaginal swab samples.