Objectives: The study aimed to determine the incidence of infections caused by M. hominis and U. urealyticum at the University Hospital of Split from 1.1.2020 to 31.12.2020 and to determine the susceptibility of these isolates to antibiotics in the same period. Materials and methods: The study was carried out on data on patients with isolated M. hominis or U. urealyticum during this period. The data were collected from the Department of Microbiology and Parasitology, University Hospital of Split (UHS). As a diagnostic method, the commercial cultivation set MYCOFAST RevolutioN was used, divided into two phases of the test. The screening phase examines the presence (positive result) or absence (negative result) of mycoplasma and ureaplasma in the sample. The second phase is carried out on samples that have been proven mycoplasma or ureaplasma in the screening test, and its purpose is to identify and quantify the isolated bacterium and to examine its susceptibility to antibiotics. The principle of both phases of the test is similar, the wells of the sets are filled with a liquid medium containing the nutrients necessary for the growth of both bacteria, specific antibiotics with which it is possible to identify or determine the sensitivity of bacteria, and an indicator that visualizes the increase of bacteria in the well. Results: Department of Microbiology and Parasitology UHS received 638 samples suspected of infection with ureaplasma and mycoplasma. Of these, 590 samples were negative, and in 7% (45 samples) U. urealyticum and 0.5% (3 samples) M. hominis were proven. Ureaplasma was predominantly isolated from the genital system in women of generative age. The majority of tested strains were susceptible to doxycycline (94,4 %), erythromycin (82,9 %), moxifloxacin (80,5 %), levofloxacin (77,8 %) and tigecycline (76 %); and resistant to clindamycin (95,8 %). However, the sample size for mycoplasma was too small for statistical analysis.