The aim of present study was to evaluate the occurrence of Chlamydia trachomatis, Mycoplasma hominis, and Ureaplasma urealyticum in non-gonococcal urethritis (NGU) and to determine the bacterial resistance to six antibiotics in order to determine the most suitable treatment strategy. A total of 50 patients were enrolled into the study. Urethral samples were taken with a dacron swab placed into urethra 2-3 cm in males, and vaginal samples were taken from the endocervical region in women. The patient samples that did not grow Neisseria gonorrhoeae were accepted as NGU. Direct immunofluorescence technique was used for the investigation of C. trachomatis. Mycoplasma IST was used for the isolation of M. hominis and U. urealyticum. U. urealyticum was isolated from 24 patients. Thirteen of them had only U. urealyticum, and the rest had mixed pathogen organisms