In <10% of patients with prostatitis syndrome, a causative uropathogenic organism can be detected. It has been shown that certain organisms that cause sexually transmitted infections can also cause chronic bacterial prostatitis, which can be hard to diagnose and treat appropriately because prostatic samples obtained by prostatic massage are not routinely tested to detect them. We conducted a clinical study to determine the prevalence of Chlamydia, mycoplasma, and trichomonas infection in 254 patients that were previously diagnosed and treated for chronic prostatitis/chronic pelvic pain syndrome due to negative urethral swab, urine, and prostate samples. Urethral swabs and standard Meares–Stamey four-glass tests were done. Detailed microbiological analysis was conducted to detect the above organisms. Thirty-five (13.8%) patients had positive expressed prostatic secretions/VB3 samples, of which 22 (10.1%) were sexually transmitted organisms that were not detected on previous tests.