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This study aimed to assess the colonization prevalence and antibiotic susceptibility of genital Ureaplasma urealyticum and Mycoplasma hominis in a teaching hospital, in Turkey. A total of 382 sexually active women with abnormal vaginal discharge were included in the study. Samples that were obtained with cotton swabs were microbiologically analyzed for U. urealyticum and M. hominis, together with antimicrobial susceptibility to doxycycline, ciprofloxacin, ofloxacin, erythromycin, josamycin, pristinamycin, and tetracycline. Ureaplasma urealyticum was detected in 185 (48.4%) cultures, and M. hominis in 17 (4.4%). Eight (2.1%) cultures were positive for both. Resistance of M. hominis to doxycycline, ciprofloxacin, ofloxacin, erytromycin, josamycin, pristinamycin and tetrascycline was 5.9%, 17.6%, 41.2%, 88.2%, 5.9%, 5.9% and 11.8%, respectively. Resistance to doxycycline, ciprofloxacin, ofloxacin, erytromycin, josamycin, pristinamycin and tetrascycline in U. urealyticum isolates was 1.6%, 40.5%, 58.4%, 54.0%, 1.6%, 8.1% and 13.5%, respectively. Both U. urealyticum (94.1%) and M. hominis (96.2) were most sensitive to josamycin, and most resistant to erytromycin (U. urealyticum 54.0%, M. hominis 88.2) and ofloxacin (U. urealyticum 58.4%, M. hominis 41.2%). As a result, the rate of U. urealyticum and M. hominis was found to be 48.4% and 4.4%, respectively. We conclude that doxycycline may be used in empirical treatment of genital tract infections in sexually active women.