Objectives: The aim of the study was to examine the frequency of urogenital mycoplasma infections in the studied group and to determine if there are differences in frequency according to age and gender. Additionally, their antimicrobial susceptibility was tested. Study design: Cross-sectional study with historical data. Participants and methods: The study used patient results obtained from the database of the Clinical Microbiology and Hospital Infections Department at the Clinic for Infectology of KBC Osijek from 18.05.2020. to 31.12.2023. The samples were processed using „Mycoview ID Quantum“ test, which is based on the specific metabolic properties and intrinsic resistance of mycoplasmas. This test is used for identification, titration, and resistance testing of urogenital mycoplasmas. The presence of mycoplasmas, as well as their antimicrobial susceptibility, is determined by the change in the medium color from yellow to pink. Results: A total of 1245 patients and 1367 samples were received and processed. In total, 2811 analyses were performed, resulting in the isolation of 483 M. hominis and Ureaplasma spp. isolates. Out of the total number of patients, 434 tested positive. According to the gender distribution, the majority of positive subjects were women (97,2 %). By age group, the highest number of positive subjects was between 21 and 40 years old. Among the isolates, Ureaplasma spp. was isolated in 419 (86,7 %) samples, and M. hominis in 64 (13,3 %) samples. Conclusion: The frequency of Ureaplasma spp. is higher than that of M. hominis in the studied group. The infection most commonly affects individuals between the ages of 11 and 50, especially those between 21 and 40 years old, and is more common in women. M. hominis is susceptible to doxycicline, josamycin, and ofloxacin, but resistant to azithromycin and erithromycin, while Ureaplasma spp. is susceptible to azithromycin but resistant to clindamycin and ciprofloxacin.