The present study was conducted to detect the prevalence of C. trachomatisand genital mycoplasmas; namely U. urealyticum, and M. hominis, as causes ofcervicitis and to compare different laboratory methods for the diagnosis of theseorganisms. Endocervical swab specimens were collected from 50 sexually active nonpregnantwomen in the reproductive period, with mean age of 32.1 ± 7.5 years,presenting with abnormal vaginal discharge or having signs of cervicitis onexamination in the Gynecology outpatient clinic of Cairo University Hospitals. C.trachomatis was detected by real-time PCR assay (LightMix® kit) in 3 patients (6%),while it was not detected by DFA in any of the samples. U. urealyticum was detectedby Mycoplasma IST2 in 28 cases (56%), while it was detected by in-house simplexand multiplex PCR in 8 cases (16%) and 5 cases (10%), respectively. M. hominis wasdetected by Mycoplasma IST2 in 12 cases (24%), while it was detected by bothsimplex and multiplex PCR in 10 cases (20%). There was no association between age,parity, methods of contraception used, any of the presenting symptoms, the presenceof cervical signs or the presence of pus cells in endocervical smears and any of thetested micro-organisms. It is concluded that Mycoplasma IST2 culture is moresensitive for detection of genital mycoplasmas whereas real-time PCR is a betteroption for detection of C. trachomatis.