Biofilms are species rich, partially due to highly effective powers of diffusion of the microorganisms and have wide tolerance to marine environmental conditions. Characteristically, the first organisms to respond to and convalesce from stress. The present study aims to isolate and identify the biofilm forming bacterial species, collected from surface water and substratum of the ship hull for four seasons at Chinnamuttom fishing harbor, Southeast coast of India, during June 2015 to May 2016. Among the mean concentration of bacterial isolates of both water and substratum of the station, maximum in monsoon and minimum in summer seasons. Totally 16 isolates were obtained, based on the adherence property, 8 isolates from surface water and 8 isolates from substratum of the ship hull. The samples were plated on Zobell marine agar medium for bacterial isolates of study area. The isolates of Bacillus sp., Flavobacterium sp., Pseudomonas sp., Aeromonas sp., Micrococcus sp., Vibrio sp., Salmonella sp., Staphylococcus sp., Shegella sp., Klebsiella sp., Corynebacterium sp., Enterobacter sp., Chromohalobacter sp., Bacillus sp., Escherichia coli and Bacilus sp. were reported in all the seasons at study area. The major diverse bacterial isolates were further characterized through morphological and molecular identification. Based on 16S rRNA gene sequences. Biofilm bacterial isolates were confirmed as Bacillus sp., and Pseudomonas sp.