The purpose of this study was to determine the distribution of gram-negative bacteria isolated from companion animals with sepsis, and to investigate the antimicrobial susceptibility patterns of the isolates. Bacterial pathogens were isolated from specimens of dogs and cats submitted to National Veterinary Research and Quarantine Service between 2008 and 2009. A total of 44 gram-negative pathogens were isolated from necropsied organs. The most common isolates were E. coli (n = 33), K. pneumoniae (n = 4) and B. bronchiseptica (n = 4). Most of gram-negative isolates were susceptible to ceftiofur (68.2%), colistin (84.1%), florfenicol (84.1%) and spectinomycin (61.4%). Most of those were resistant to ampicillin (77.3%), erythromycin (86.4%), flumequine (65.9%), lincomycin (97.7%), oxytetracycline (61.4%), penicillin (100%), streptomycin (63.6%), spiramycin (97.7%), sulfamethoxazole (90.9%), tylosin (97.7%) and tiamulin (100%). In conclusion, colistin and florfenicol could be useful against sepsis due to gram-negative bacteria.