Biosurfactants are fascinating bioproducts because they are amphiphilic molecules with a wide range of functions, environmental acceptability and have several industrial applications. Due to non-toxicity, biodegradability and costeffectiveness, biosurfactants are considered as excellent substitute to the chemically synthesized surfactants. The aim of present study is isolation and characterization of novel potent biosurfactant producer bacterial strains of water habitat. At sea port Karachi, ten water samples were collected from two different oil spill water points. After isolation and enrichment of the bacterial communities, they were screened for the biosurfactants production by employing blood hemolysis, drop collapse assay, oil spreading technique, and emulsification activity. All bacterial strains from oil-contaminated environment showed highest biosurfactants production potential. Bacillus wiedimanii (MWS5) was identified as the top biosurfactant producer by morphological, biochemical, and 16S rRNA techniques. Lipopeptide type biosurfactant was confirmed by Fourier transform infrared spectroscopy (FTIR) and thin-layer chromatography (TLC). The extracted biosurfactant showed antibacterial potential against different pathogenic bacterial strains.