Aims: This study aimed to detect bacterial pathogens that cause sexually transmitted diseases (STD) using multiplex polymerase chain reaction and reverse hybridization. Methodology and results: Thirty urine samples were collected from male patients aged between 20 and 45 in Dohuk City who were suspected of having an STD. The samples were tested for the presence of five main types of bacteria, namely Ureaplasma urealyticum, Neisseria gonorrhoeae, Mycoplasma hominis, Mycoplasma genitalium and Chlamydia trachomatis responsible for causing STDs. Nineteen of the thirty urine samples were positive for at least one of the five species of bacteria, yielding a positive rate of 63.3%. Ureaplasma urealyticum had the highest diagnostic rate of 68.4% among positive samples, while C. trachomatis had the lowest diagnosis rate of 5.2%. Both N. gonorrhoeae and M. hominis had a 15.7% diagnosis rate, while M. genitalium had a 10.5% diagnosis rate. Conclusion, significance and impact of study Research findings suggest that U. urealyticum was the most common cause of STD, accounting for 68.4% of the positive samples. Conversely, the study identifies C. trachomatis as the least prevalent cause, accounting for only 5.2% of the cases. These noteworthy findings shed light on the prevalence of these bacterial pathogens in sexually transmitted diseases, laying the groundwork for more precise and effective diagnostic and treatment options.