A gene encoding lipase enzyme from Bacillus flexus PU2 was cloned and expressed in E. coli BL21 (DE3) pLysS and purified protein having the molecular weight of 34 kDa. This lipase was found to be alkaline (pH 9) and slightly thermophilic. This lipase was observed to retain its activity in the presence of methanol, ethanol, DMSO, and acetone. Ferrous sulfate, copper sulfate, and manganese sulfate highly enhanced the lipase activity. All the surfactants and detergents were found to inhibit the enzyme activity, whereas the bleaching agent hydrogen peroxide was found to increase the activity. This lipase was observed as a metalloenzyme, and its activity was highly inhibited by EDTA. Also, it is moderately halophilic and can retain the activity between 0.2 and 0.8 M NaCl. Biofilm inhibitory potential of purified lipase was tested against pathogenic Vibrio parahaemolyticus, and the minimal inhibitory concentration observed was 350 U. Different concentration of this enzyme significantly changed the morphology and biofilm density of V. parahaemolyticus and was evinced by SEM and CLSM imaging. The transcriptome levels of genes responsible for biofilm formation, motility, and virulence such as, motX, fliG, and trh were significantly downregulated with lipase treatment.