The use of PCR assays as a fast and reliable method is constantly improving and easing microbiological diagnosis. We used a polymerase chain reaction (PCR) assay designed to detect Mycoplasma genitalium and Chlamydia trachomatis in urethral swab samples of 56 males with urethritis and 44 asymptomatic patients as a control group. The PCR assay provides an amplification of target sequence within MgPa (M. genitalium protein attachment) gene. Results indicated that M. genitalium was present in 6 (10.7%) patients with urethritis and none in the control group. Eleven of 56 (17.8%) patients were positive for Chlamydia trachomatis when tested by an outer membrane protein primer-based PCR. The amplified DNA fragments were homogeneous as shown by restriction enzyme analysis and found to be consistent with the published sequences. The PCR assay employed was as reliable as the cultural method in detecting C. trachomatis in the urethral swabs of patients with urethritis (100% of sensitivity when compared with the cultural method) and it has been revealed as an essential method for detection of M. genitalium.