Compost generated through fish waste composting could provide an effective source of nutrient-rich organic matter for microbial growth, leading to the production of valuable products such as biosurfactants. Existing biosurfactant production is a relative expensive process and raw materials contribute about 30% of the production cost. Utilizing waste streams such as fish waste compost (FWC) as a substrate is an economically viable alternative. In this study, biosurfactant was produced by Rhodococcus erythropolis sp. P6-4P, a strain isolated from the North Atlantic Ocean. Biosurfactant production with FWC extract was compared with other soluble and insoluble carbon and nitrogen sources using emulsification assay and surface tension measurement. FWC extract showed good potential as an unconventional source of nutrient for microbial growth. The produced biosurfactant under optimum condition obtained via response surface methodology was further characterized for total carbohydrate, total lipid and total protein content. The results provided evidence for using FWC extract as a novel substrate for biosurfactant production