Sexually transmitted diseases (STDs) are a very important cause of illness worldwide and prolonged, untreated infections with STD pathogens may have serious consequences. Our study aims to evaluate the distribution of six different STDs (Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Ureaplasma urealyticum, Mycoplasma hominis and Mycoplasma genitalium) in male urine samples. First void urine samples from 52 symptomatic patients were collected between April 2014 and April 2015. DNA was extracted, purified and amplified via multiplex polymerase chain reaction (PCR) for the detection of the six STD pathogens, further identified using a 2% agarose gel electrophoresis with ethidium bromide as staining agent. STD frequency in the study group was 53.84 % (28 patients), mostly in the 20-29 years age group. Among positive patients, six presented multiple infections. 35 positive DNA samples were identified in the study: 17 of C. trachomatis, 9 of U. urealyticum, 7 of N. gonorrhoeae and 2 of M. genitalium. Wide scale application of the system based on the simultaneous detection of these six pathogens inducing STD may facilitate diagnosis, especially in multiple infections.