The aim of our study was to determine the antimicrobial susceptibility profiles of 267 Gram-negative clinically significant anaerobes, isolated between October 2016 and October 2019, in a Greek university hospital. The species identification was performed by conventional methods and using the Vitek 2 automated system. Antimicrobial susceptibility testing to determine the MICs was performed by the E-test method. The antimicrobial agents tested were penicillin, ampicillin, amoxicillin-clavulanic acid, piperacillin-tazobactam, cefoxitin, imipenem, meropenem, clindamycin, metronidazole, moxifloxacin, chloramphenicol and tigecycline. The results were interpreted using the CLSI and FDA breakpoints. The majority of the isolates belonged to Bacteroides fragilis group (58.8%), followed by Prevotella spp. (23.2%), Fusobacterium spp. (11.2%) and Veillonella spp. (6.4%). The most prevalent types of infection were skin and soft tissue infections (34.8%), and inta-abdomonal infections (29.6%). Among all isolates tested, the lowest rates of resistance (<5%) were detected to carbapenems, metronidazole, chloramphenicol and tigecycline. Resistance to piperacillin-tazobactam was observed in 5.4%, 24.6%, 3.3% and 17.6%, of B. fragilis, B. fragilis group, Fusobacterium spp. and Veillonella spp. isolates, respectively. Although a high prevalence of resistance to clindamycin, cefoxitin, and moxifloxacin, was detected particularly among members of the B. fragilis group, cefoxitin resistance was low for Prevotella spp. (3.2%), Fusobacterium spp. (3.3%) and Veillonella spp. (0%). Our findings underscore the need for periodic monitoring of antimicrobial resistance in order to guide empirical therapy. (C) 2020 Elsevier Ltd. All rights reserved.