The purpose of this study was three fold: to compare semen and first void urine (FVU) specimens from asymptomatic infertile men for the detection of Chlamydia trachomatis, genital ureaplasmas, and mycoplasmas infections using in-house inhibitor-controlled PCR-microtiter plate hybridization assay, to determine the prevalence of those organisms in infertile men in Tunisia and to study the relationship between these bacteria and male infertility. Paired urine and semen specimens from 104 patients were examined by the in-house PCR for the presence of Chlamydia trachomatis, genital ureaplasmas (Ureaplasma urealyticum and Ureaplasma parvum), and mycoplasmas (Mycoplasmas hominis and Mycoplasma genitalium) DNA. Semen analysis was assessed according to the guidelines of the World Health Organization. Nominal scale variables, Mann-Whitney test and Kruskal-Wallis Nonparametric ANOVA test were used for statistical analysis. There was a very high concordance (> 95 %) and a very good agreement (K > 0.9) between the detection of Chlamydia trachomatis, genital ureaplasmas, and Mycoplasmas hominis in semen and corresponding FVU specimens. Our findings also shows, a high concordance (81.1 %) and a good agreement (K=0.79) between the detection of Mycoplasma genitalium in both specimens. Chlamydia trachomatis, genital mycoplasmas, and genital ureaplasmas were found to be widespread among infertile male patients in Tunisia, as shown by their respective prevalences 43.3%, 18.3%, and 14.4%. The mean values of seminal volume, sperm concentration, sperm viability, sperm motility, sperm morphology, and leukocyte count were not significantly related to either the detection of Chlamydia trachomatis DNA or to that of genital ureaplasmas and mycoplasmas DNA in semen specimens. Using our in-house PCR, both semen and FVU were found to be sensitive diagnostic specimens for the detection of Chlamydia trachomatis, ureaplasmas, and mycolasmas. The FVU, a less invasive and self-collected specimen, can serve as a marker for the presence of these organisms in the genital tract and can be used as a reliable way of detecting asymptomatic carriers of infection.