Biosurfactants are a group of microbial molecules identified by their unique capabilities to interact with hydrocarbons. Emulsification and de-emulsification, dispersion, foaming, wetting and coating are some of the numerous surface activities that biosurfactants can achieve when applied within systems such as immiscible liquid/liquid (e.g., oil/water), solid/ liquid (e.g., rock/oil and rock/water) and gas/liquid. Therefore, the possibilities of exploiting these bioproducts in oil-related sciences are vast and made petroleum industry their largest possible market at present. The role of biosurfactants in enhancing oil recovery from reservoirs is certainly the best known; however they can be effectively applied in many other fields from transportation of crude oil in pipeline to the clean-up of oil storage tanks and even manufacturing of fine petrochemicals. When properly used, biosurfactants are comparable to traditional chemical analogues in terms of performances and offer advantages with regard to environment protection/conservation. This chapter aims at providing an up-to-date overview of biosurfactant roles, applications and possible future uses related to petroleum industry.