Contamination of water and soil with crude oil and petroleum compounds frequently occurs as the result of accidental spills and leaks and causes environmental contamination that is either difficult to be removed by traditional remediation techniques or is not economically feasible. The development of effective and feasible processes and technologies that can decrease the damaging effects of oil contamination and lower treatment costs are of great importance. Given the high surface and interfacial activities, temperature and ionic tolerance, biodegradability and low toxicity of biosurfactants (BSs)/bioemulsifiers (BEs) and the fact that numerous BSs/BEs-producing microorganisms have been isolated from oil-contaminated environments, the applications of these compounds have been investigated for emulsification, solubilization, corrosion inhibition, viscosity reduction, and biodegradation of oil. To date, full-scale applications of BSs/BEs in the oil industry have not yet achieved widespread recognition, thus the aim of this chapter is to review the successful examples of laboratory, field-scale and full-scale applications of BSs/BEs in oil activities and cleanup of petroleum contaminated environments to encourage the oil industry and decision makers to consider the utilization of BSs/BEs for such activities.