Bacteriophage therapy is one of the potential and safer methods to control bacterial pathogens in the aquaculture industry recently. The present study, lytic bacteriophage was isolated from the effluent water of pacific white shrimp Litopenaeus vannamei semi intensive culture. The sample was screened against different bacterial pathogens including Bacillus cereus WPD, Vibrio harveyi, V. vulnificus, V. parahaemolyticus and V. alginolyticus for the confirmation of bacteriophages. The initial screening result revealed that, the phage possessed the strong lytic activity against V. vulnificus. There is a least activity observed against V. harveyi and V. alginolyticus. There is no lytic activity observed against B. cereus WPD and V. parahaemolyticus. The host range was performed against the same pathogens which used for the isolation process using spot test method. Further the in vivo biofilm inhibition method was performed against V. vulnificus in different concentration of lytic phage to find the better inhibition of biofilm on HPDE surface. The results revealed that, using 200 µl concentration of phage was effectively inhibit the V. vulnificus biofilm formation. The present study revealed that, the isolated phage is useful to control the V. vulnificus pathogen.