Vibrio vulnificus is a pathogen causing two types of severe illness, septicemia and wound infections, and is continually detected in marine environments. To investigate the biochemical characteristics and the antimicrobial susceptibility of V. vulnificus isolated from environment of Korea in 2001, the API 20E kit test, PCR, and antibiotic disk diffusion method were performed. A total of 210 V. vulnificus strains was isolated from seawater, shell-fish, sediments, coastal water, aquarium water, sewage, and others. All of the isolates could be divided into 15 groups on the basis of their API 20E profiles, and were positive in Indole test. Only 173 isolates (82.4%) were positive by the PCR amplifying the cytolysin-hemolysin gene. Almost all isolates were susceptible to chloramphenicol (99.5%), tetracycline (90.0%), ciprofloxacin (92.4%), trimethoprim/sulfamethoxazole (89.0%), nalidixic acid (87.6%). Some isolates were resistant to cephalothin (57.6%), amikacin (33.3%), cefoxitin (31.9%). One hundred and forty three isolates (68.1%) were resistant to one or more antimicrobial agents. These results show that V. vulnificus environmental isolates possessed various biochemical characteristics, and some isolates were not detected of the cytolysin-hemolysin gene by PCR, and a part of isolates were resistant to multiple antibiotics.