Chlamydia trachomatis and Neisseria gonorrhoeae are important bacterial pathogens of sexually transmitted infections (STIs) worldwide. This study sought to compare the analytical sensitivity and specificity of conventional methods against a rapid molecular method in detecting STIs caused by these bacteria. Methods: Ninety five first-time male attendees of the Genito-urinary Medicine Clinic in Hospital Kuala Lumpur were included in this cross-sectional study. The detection of C. trachomatis was achieved through direct fluorescence antibody (DFA) staining of urethral swabs and real-time polymerase chain reaction testing (Xpert® CT/NG assay) on urine specimens. N. gonorrhoeae was detected through Gram staining and culture of urethral swabs and Xpert® CT/ NG assay on urine specimens. Results: From the Xpert® CT/NG results, 11 (11.6%) attendees had chlamydia, 23 (24.2%) had gonorrhoea and 8 (8.4%) had both STIs. The sensitivity and specificity of DFA in detecting chlamydia compared to Xpert® CT/NG were 5.3% (95% CI: 0-28) and 94.7% (95% CI: 86-98), respectively. For gonorrhoea, the sensitivity and specificity of Gram staining were 90.3% (95% CI: 73-98) and 95.3% (86-99), respectively, whereas the sensitivity and specificity of culture compared to Xpert® CT/NG were 32.2% (95% CI: 17-51) and 100% (95% CI: 93-100), respectively. Conclusion: Although Gram-stained urethral swab smears are sensitive enough to be retained as a screening tool for gonorrhoea, culture as well as DFA lack sensitivity and are poorly suited to screen for gonorrhoea and chlamydia, respectively. However, owing to their high specificity, conventional detection methods are still suitable as confirmatory tests for gonorrhoea and chlamydia.