Background: The aim of this study was to assess the presence of M. hominis and U. urealyticum agents, their distribution between male and female, and differences in antibiotic susceptibility in samples sent from Hisar Intercontinental Hospital's various clinics with the preliminary diagnosis of genitourinary system infection. Methods: The Mycoplasma IES test was used to identify M. hominis and U. urealyticum, and to determine antibiotic susceptibility results, in samples taken from patients. The findings of mycoplasma and ureaplasma culture testing samples requested between 2014 and 2021 were evaluated retrospectively from our records. Results: M. hominis was found to be positive in 7.37% of the examinations, U. urealyticum was found to be positive in 34.98% of the examinations, and either of them were found to be positive in 22.01% of the examinations. The growth rate of M. hominis and/or U. urealyticum was determined to be 24.95% in females and 10.13% in males, with the growth rate in females being greater and statistically significant (p < 0.001). According to the antibiotic susceptibility test results, clarithromycin (R 17.91%) was the most susceptible antibiotic overall for both microorganisms, while clindamycin (R 90.28%) was the most resistant. Depending on the sex, clarithromycin (R 18.40%) was found to be the most susceptible antibiotic in females, and levofloxacin (R 10.87%) to be the most susceptible in males. Conclusion: Given the presence of M. hominis and U. urealyticum infections, especially in the presence of risky conditions such as pregnancy, laboratory tests for the diagnosis of these agents should be used in such studies since no urogenital infections were detected in the routine cultures of the patients followed up with the suspicion of urogenital infection. Gender differences should also be considered as a parameter in the preference of antibiotics. [ABSTRACT FROM AUTHOR]