Background: Chlamydia trachomatis is the most common bacterial sexually transmitted infection in the world, but the effect of this infection on male fertility is still controversial. Despite reports of interaction between Mycoplasma genitalium and sperm, this pathogen in semen samples of infertile men is less studied. We studied, the prevalence of Chlamydia trachomatis and Mycoplasma genitalium infection in infertile men.Methods: Among attending Avicenna Infertility Center, 120 men who had abnormal semen analysis tests were selected and the samples were taken. After detailed analysis of semen quality, DNA was extracted from each sample by chelex. Samples were evaluated for these two pathogens by multiplex PCR. Results were statistically analyzed.Results: Chlamydia trachomatis and Mycoplasma genitalium was detected in 23/3% and 12/5% of the samples, respectively. Although, Mycoplasma genitalium infection rises by increasing (P=0.640) and decreasing in age of first sexually activity (P=0.203), and also positive cases of Chlamydia trachomatis infection showed increase regarding age increase (P=0.619) and age decrease in first sexually activity (P=0.511), but these differences were not statistically significant. Conclusion: All in all, regarding to the increased prevalence of Chlamydia trachomatis infection compared with the only similar study in Iran and high prevalence of Mycoplasma genitalium infection in infertile men, this assessment was done. A multiplex PCR protocol rapidly and simultaneously identify these organisms in comparison with uniplex from clinical samples. Based on our results screening for Chlamydia trachomatis and Mycoplasma genitalium infection among infertile men seems to be valuable.