The purpose of this study was to determine the β-lactamase activity and in vitro antibiotic susceptibility of clinical isolates of anaerobic bacteria. Totally, 27 anaerobic bacteria which were isolated from clinical specimens were identified with Gram reaction, colony morphology, "An-ident" disks and Vitek system. Susceptibilities of all isolates to amoxicillin/clavulanate, ciprofloxacin, cefoxitin, imipenem, clindamycin and metronidazole were determined by both agar dilution and E test methods and the results were compared. Also the β-lactamase activity of the isolates were determined by using nitrocefin sticks. Vitek system identified six of the isolates as Bacteroides uniformis, three as B. fragilis two as B. ureolyticus, two as B. ovatus, one asB. vulgatus, one as B. eggerthii, one as B. caccae two as Prevotella melaninogenica, one as P. buccae, one as P. oris, two as Clostridium perfringens, two as Clostridium tertium, two as Clostridium sporogenes and one as Peptostreptococcus anaerobius. An-ident disk identification results agreed with Vitek in 85.7% of all strains and 93.8% of Bacteroides isolates. Susceptibility rates of anaerobic bacteria to metronidazole, imipenem, amoxicillin/clavulanate, cefoxitin, clindamycin and ciprofloxacin were 100%, 96.3%, 88.9%, 81.5%, 81.5%, 48.1%, respectively. The agreement rate of MIC values by both methods was found to be 88.6% within two-fold dilutions and 95.8% when the susceptibility categories were considered. Sixteen (59.2%) of all strains and 11 (40.7%) of Bacteroides species hadβ β-lactamase activity. According to the susceptibility rates of the isolates, metronidazole and imipenem were found to be the most active agents. Also it was concluded that E test, which is practical and gives consistent results with the reference test, may be an alternative method for routine susceptibility testing of anaerobic bacteria.