Objective(s):MycoplasmaandUreaplasmaspecies threaten reproductive health and fertility worldwide. Due to the lack of sensitive, accurate, and affordable diagnostic tools, the simultaneous contributions of these agents in infertility have been overlooked. This study aims to detect and identifyMycoplasmaandUreaplasmaspecies in the genital tracts of fertile and infertile females simultaneously.Materials and Methods:In a case-control study, cervicovaginal clinical samples were collected from patients referred to two teaching hospitals in Isfahan from July 2019 to February 2019. The initial screening was by using Real-time PCR and designed primer to evaluate the presence ofMycoplasmaandUreaplasmaspecies including fertile and infertile women. The bacteria species were then detected and differentiated by using the melt curve and sequenced to confirm and identify. Finally, the standard curve was used to measure and compare the copy number of each species in each group. The isolates also were detected in clinical samples using the commercial PCR method.Results:The frequencies ofMycoplasma genitaliumandMycoplasmahominiswere (0.0, 10.0%) in the fertile group and (4.3%, 34.3%) in the infertile group, respectively.UreaplasmaparvumandUreaplasma urealyticumspecies in the fertile group (7.1%, 5.7%) and in the infertile group (32.9%, 24.3) were determined, respectively. The comparison of the results obtained from PCR and Real-time PCR showed that the recent technique has the ability to track 101–103 copy numbers.Conclusion:The present method allows differential diagnosis and quantification ofMycoplasmaandUreaplasmaspecies in a short time and simultaneously.