Abstract Biofilm are group of microbial community enclosed inside a well-organized complex structure made up of extracellular polymeric substances. Biofilm forming bacteria are the growing concern of morbidity and mortality in aquatic population. Among the different biofilm forming bacteria, Vibrio spp. are pathogenic, diverse and abundant group of aquatic organism. Aquatic animals like fish, mollusks and shrimp harbors pathogenic and non-pathogenic forms of Vibrio spp. In the present study, Vibrio strains were isolated from various region of Tamil Nadu (Chennai, Cuddalore and Tiruchirappalli) and Puducherry. A total of 155 strains were isolated from various sampling sites, among them 84 (54.7%) were confirmed as Vibrio spp. through biochemical characterization and Vibrio specific 16S rRNA gene amplification. Among the 84 strains 50 strains were confirmed as biofilm formers and they were categorized as strong (10), moderate (17) and weak (23), based on their biofilm forming ability. Further the pathogenicity of all the Vibrio strains were tested through lytic enzyme production (protease, gelatinase and hemolysin) and swarming motility. After 16S rRNA gene sequencing, the 10 strong biofilm forming strains were Vibrio alginolyticus (TFM17, TFM30, and UFM24), Vibrio parahaemolyticus (TFM1), Vibrio harveyi (CUS2), Vibrio spp. (CUS4) and Gammaproteobacteria (TFM4, TFM19, TFM61, CUS5). The present study is a first report dealing with the biofilm forming Vibrio spp. isolation from south east coastal regions of Tamil Nadu and Puducherry. Further the informations in the present study will help in finding novel strategies to combat multi drug resistance biofilm formers in aquaculture industries.