OBJECTIVES: The aim of this study was to determine the proportion of anaerobic isolates in the examined period, as well as to examine the frequency of individual bacterial species and to test the antimicrobial susceptibility of isolated anaerobic bacteria. STUDY DESIGN: A cross-sectional study. MATERIAL AND METHODS: This study analysed the results collected by testing the antimicrobial susceptibility of strictly anaerobic isolates obtained from clinical specimens in routine work at the Institue for clinical microbiology and hospital infections at Clinical hospital centre Osijek from May , 2020 until May , 2022. Solid media, like Columbia and SNVS agar were used for cultivation of anaerobic bacteria, in an antmosphere with anaerobic conditions. The identification of isolates was performed using a MALDI-TOF MS (Bruker, Ultraflex) device. Antibiotic susceptibility measurements were performed by the breakpoint method - commercially available test (ATB AN System). RESULTS: There were 6693 samples with a request for anaerobic analysis and 556 samples were positive after anaerobic incubation. The largest number of pathogens, 283 (46,78 %), were isolated from blood cultures and cerebrospinal fluid. Strict anaerobes were mostly isolated from the skin, subcutaneous and intraoperative samples. Among the isolated genera, genus Bacteroides spp. with 130 (47,45 %) isolates is the most dominant. The most frequently isolated species is Bacteroides fragilis with 71 isolates. There are 186 (80,17 %) isolates of gram-negative anaerobes that are showing resistance to penicillin and 227 (97,84 %) isolates that are susceptible to metronidazole. Gram-positive anaerobes are susceptible to the tested antibiotics in a high percentage from 61,54 % to 100,00 %. 8. Summary 28 CONCLUSION: The results of this research show the importance of antimicrobial susceptibility testing in everyday laboratory practice, as well as the importance of monitoring antimicrobial resistance. Metronidazole is the first-line treatment for infections caused by anaerobes.