The present studies highlight the isolation, screening, production and evaluation of bioremediation efficacy of marine microbial surface-active agent on crude oil contaminations. Based upon screening, six isolates were confirmed as capable of producing surface active agents were obtained from marine sediments of Ennore harbor, Chennai, Tamil Nadu and these isolates were identified as bacillus genera with significant differences in their morphology. For the maximum production of surface-active agents, nutritional optimization in 6 screened isolates revealed that sucrose and yeast extract were the suitable carbon and nitrogen sources for growth in pH of 7.2 ± 0.2, at 37°C temperature and agitation at 180-200 rpm. Extraction and characterization studies revealed, the product was polymeric in nature with surface activity (28 ± 4 mN/m), thermal and emulsification stability (more than 90 days) when compared with synthetic surfactants. Based on laboratory scale studies, 90% of crude oil was removed from the aqueous phase within 60-120 minutes of exposure to the partially purified surface-active agent. This study suggested that the Bacillus species can be utilized for cleaning up hydrocarbon contamination sites and concurrently produce bio surfactants.