Clostridial bacteria are zoonotic agents, which cause severe necrotizing enteritis, pseudo-membrane colitis, enterotoxemia to both humans and animals. The objective of this study was to monitor the antibiotic resistance of Clostridium isolates on clinical specimens from wild animals in Seoul zoo for 5 years. Clostridium isolates were verified by using Vitek2 compact machine. Antibiotic susceptibility was assessed by antibiotic disc diffusion test, which was followed by Kirby-Bauer disc diffusion test method. The frequency of Antimicrobial resistance of Clostridium isolate was the greatest in gentamicin (87%), then in order of amikacin (80%). There were 55.6% of Clostridium isolates showed multiple drug resistance (MDR). These results showed that a lot of Clostridial bacteria from wild animals in Seoul zoo were acquired antibiotic resistance. Because of the wild animal’s aggressive manner, it has been hard to collect clinical samples from wild animals in a zoo to exam antibiotic susceptibility. For these reasons, empirical use of antibiotics has been performed in frequently. It may cause to increase the emergence of antibiotic resistance bacteria. In addition, the antibiotic resistance bacteria from zoo animals can be spread to other wild animals which inhabit around the zoo. Therefore, regular monitoring of antibiotic resistance Clostridial bacteria is important to protect animals and humans from Clostridial diseases.