Vibrio cholerae, causative agent of profuse watery diarrhea forms biofilm structure which is adaptive advantage for the Vibrio cholerae. This study was carried out to characterize certain Vibrio cholerae environmental strains collected from the water reservoirs surrounding Midnapore town, West Bengal, India in respect of serogroups, antibiotic susceptibility, protease activity, haemolytic activity, pathogenecity and determination of biofilm forming ability and also signaling system involved in the regulation of biofilm. Multiplex PCR assay revealed that all were from non O1/non O139 serogroups and ctxA, tcpA negative. All of them were sensitive to streptomycin, tetracycline, nalidixic acid but resistant to ampicillin, norfloxacin and chloramphenical and 70% were polymyxin B resistant. Beside this these Vibrio cholerae isolates were strongly hemolytic, moderate to high biofilm forming. In this study we found that these strains did not follow the biofilm controlling previously reported flagella-mediated, hapRmediated as well as quorum sensing autoinducer(s)-mediated signal transduction pathway.