Background and Objectives: Production and use of valuable compounds such as biosurfactants from wastewater, with regard to its environmental and economic benefits, is of particular interest recently. Nowadays, biosurfactants produced by microorganisms are used in various food sources, pharmaceuticals, petrochemicals, oil extraction, and wastewater treatment plants. This study was conducted to evaluate the use of oily wastewater as a cheap substrate using Pseudomonous aeruginosa strains isolated from hospital wastewater to produce rhamnolipid biosurfactants.Materials and Methods: The present study was an experimental study that was conducted in a laboratory scale in a batch reactor containing local bacteria isolated from hospital sewages wastewater in a mineral medium containing various oils so that the culture oil was evaluated at different times and concentrations, using hemolysis, oil spill, droplet removal, emulsifying activity, and chemical oxygen demand experiments. In this study, the statistical analyses were performed based on the assessments of the control-test groups according to student’s t-test.Results: The obtained results showed that Pseudomonas aeruginosa bacteria had a good ability to produce biosurfactant. Reduced surface tension, more than 70% emulsification, and 85% COD (Chemical Oxygen Demand) reduction were the results of using isolated biosurfactant.Conclusion: Based on the findings of this study, it seems that oily sewages containing high amounts of organic matters are the suitable option for producing rhamnolipid biosurfactants. Produced biosurfactants, due to the desired emulsification properties, can be used in elimination of decomposable pollutants and biodegradation processes.