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<i>Chlamydia trachomatis, Neisseria gonorrhoeae</i>, and <i>Mycoplasma genitalium</i> are the three most commonly reported sexually transmitted bacteria. The present study aimed to investigate the presence of <i>C. trachomatis, N. gonorrhoeae,</i> and <i>M. genitalium</i> in urogenital samples collected from 18–68-year-old Turkish patients who were admitted to the hospital with various urogenital symptoms. A total of 360 patients with symptoms of STD were included in the study. Following DNA extraction by QIAamp Mini Kit, the presence of <i>C. trachomatis, N. gonorrhoeae,</i> and <i>M. genitalium</i> were investigated using multiplex real-time PCR. Causative organisms were identified in 68 (18.9%) of 360 patients. <i>C. trachomatis, N. gonorrhoeae</i>, and <i>M. genitalium</i> were detected in 40 (11.1%), 14 (3.9%), and 28 (7.8%) of the patients, respectively. Patients 21–30 years of age represented more than one-third (37.8%) of positive patients. Of all patients, dual infections of <i>C. trachomatis–M. genitalium, N. gonorrhoeae–C. trachomatis, N. gonorrhoeae–M. genitalium,</i> and triple infection of <i>C. trachomatis–N. gonorrhoeae–M. genitalium</i> were determined in 1.6% (6/360), 1.3% (5/360), 0.2% (1/360), and 0.2% (1/360) of the patients, respectively. In CT-, NG-, and MG-positive patients, different STI agents were also found such as HIV, HBV, HPV, HSV2, T. pallidum, and T. vaginalis. In conclusion, among <i>C. trachomatis, N. gonorrhoeae,</i> and <i>M. genitalium</i>, CT was the most frequently detected bacterial cause of STDs in our hospital at Istanbul. Co-infections, which comprise more than one-fifth of the cases, should not be underestimated. Regular screening and following up of STD agents using multiplex real-time PCR-based diagnostic methods enabling the immediate detection of co-infections are essential for the treatment and primary prevention of STDs.