The aim of this study was to identify gram-negative anaerobic bacilli isolated from various clinical specimens that were obtained from patients with suspected anaerobic infections and to determine the antibiotic resistance profiles by using the antibiotic concentration gradient method. The study was performed in Afyon Kocatepe University Ahmet Necdet Sezer Research and Practice Hospital, Medical Microbiology Laboratory between 1 November 2014 and 30 October 2015. Two hundred and seventyeight clinical specimens accepted for anaerobic culture were enrolled in the study. All the samples were cultivated anaerobically by using Schaedler agar with 5% defibrinated sheep blood and Schaedler broth. The isolated anaerobic gram-negative bacilli were identified by using both the conventional methods and automated identification system (VITEK 2, bioMerieux, France). Antibiotic susceptibility tests were performed with antibiotic concentration gradient method (E-test, bioMerieux, France); against penicillin G, clindamycin, cefoxitin, metronidazole, moxifloxacin, imipenem, meropenem, ertapenem and doripenem for each isolate. Of the 28 isolated anaerobic gram-negative bacilli; 14 were identified as Bacteroides fragilis group, 9 were Prevotella spp., and 5 were Fusobacterium spp. The highest resistance rate was found against penicillin (78.5%) and resistance rates against clindamycin and cefoxitin were found as 17.8% and 21.4%, respectively. No resistance was found against metronidazole, moxifloxacin, imipenem, meropenem, ertapenem and doripenem. As a result, isolation and identification of anaerobic bacteria are difficult, time-consuming and more expensive when compared with the cost of aerobic culture. The rate of anaerobic bacteria isolation may be increased by obtaining the appropriate clinical specimen and appropriate transportation of these specimens. We believe that the data obtained from the study in our center may offer benefits for the follow up and treatment of infections caused by anaerobic bacteria and may contribute to the current literature. Because of high resistance rate detected against penicillin, this antibiotic should not be used as a first choice in empirical treatment. Cefoxitin may be used in empirical antimicrobial treatment of anaerobic gram-negatives; but the rate of antibiotic resistance should be detected for more useful and proper treatment. The prior selection of the most effective antibiotic, may contribute to decrease the rate of high resistance. In our study, no resistance was observed against carbapenem group antibiotics and metronidazole; so these antibiotics should be reserved as treatment options in the future for infections caused by resistant gram-negative anaerobic bacteria.