The principal objective of this study was to determine the optimal conditions for the production of biosurfactant by the indigenous bacterium, Pseudoalteromonas sp. HK-3, originating from oil-spilled areas. The relationship between total biosurfactant production and the factors affecting biosurfactant production were evaluated by statistical analysis using SPSS software. The effects of various supplemental carbon sources (e.g., glucose, dextrose, mannitol, citrate, acetate) on the maximal production of biosurfactant by the test culture of Pseudoalteromonas sp. HK-3 was then evaluated. As a result, mannitol was found in this study to be the best supplemental carbon source for the production of biosurfactant. A spot inoculation of crude cultural liquid containing the HK-3 cells generated the largest clear zone, whereas only small clear zones appeared around the spots inoculated with either supernatant only or cell pellets following centrifugation. Our results demonstrated that the HK-3 test culture supplemented with 2% mannitol at an initial pH of 6 generated the maximal amount of biosurfactant within 72 h of incubation.