In view of the increasing antibiotic resistance of anaerobic Gram-negative bacteria, we determined the antimicrobial profile of 55 periodontal anaerobic Gram-negative bacteria correlated with human infections, comprising 16 strains of Fusobacterium nucleatum and 39 strains of Prevotella spp. isolated from periodontal pockets of 26 adults suffering from chronic periodontitis. All the strains of F. nucleatum were susceptible to amoxicillin/clavulanic acid, doxycycline, metronidazole, moxifloxacin and levofloxacin, whilst 2/16 strains were both resistant to amoxicillin and beta-lactamase-positive and 11/16 were resistant to clarithromycin. All of the Prevotella strains were susceptible to amoxicillin/clavulanic acid, doxycycline and metronidazole, whereas 7/39 strains were beta-lactamase-positive and resistant to amoxicillin, 5/39 were resistant to clarithromycin and 3/39 were resistant to both moxifloxacin and levofloxacin. Our findings confirm that there is an increasing need to encourage practitioners to use laboratory investigations to limit the risk of an incorrect therapeutic approach and to avoid the overuse of antimicrobial agents.