OBJECTIVES: The aim of this study to determine frequency of specific anaerobic isolates during the specified period, assess the antimicrobial suspectibility of the isolates using disk diffusion and breakpoint method, and compare the results between the two methods. STUDY DESIGN: Cross–sectional study. MATETIAL AND METHODS: This study analysed results obtained by comparing antimicrobial suseptibility testing using the breakpoint method and the disk diffusion method for strictly anaerobic isolates from samples collected during routine work at the Institue for clinical microbiology and hospital infections at Clinical hospital centre Osijek from June 1, 2022, to May 31, 2023. Culture media, such as Columbia agar and Schedler Neomycin Vancomycin agar (SNVS), were used for cultivation of anaerobic bacteria, along with thioglycolate broth as needed. The identification of isolates was performed using a MALDI-TOF MS (Bruker, Ultraflex) device. Suspectibility testing of isolates was performed using breakpoint method with a commercially available system (ATB ANA, Biomerieux, France) and the disk diffusion method on fastidious anaerobic agar (FAA). RESULTS: Out of a total of 511 (7.05 %) anaerobic isolates, 150 (29.35 %) were stictly anaerobic bacteria. The incidence od strictly anaerobic bacteria was 2.07 %. The study included 114 samples, with the Bacteroides spp. genus being most common, accounting for 61 (51.51 %) isolates, while the Clostridium spp. genus had the lowest number of samples, with 4 (3.51 %) isolates. Susceptibility testing using the disk diffusion and breakpoint method indicated that the most effective treatments for anaerobic isolates were tazobactam/piperacilin, carbapenems and metronidazole, while penicilin and clyndamycin showed the least effectivness. CONCLUSION: The results of this study demonstrate the importance of antimicrobial susceptibility testing and adherence to appropriate interpretation criteria. It enables the proper selection of antibiotics for infection treatment, reduces the risk of antibiotic resistance and ensures personalized and effective patient care.