Petroleum hydrocarbons have caused environmental pollution problems at many sites in Egypt such as Nile River, Red sea and petroleum companies. Biosurfactants are promising compounds for cleaning up this contamination because of its ability to increase solubility of oil in water. Biosurfactants are friendly to environment as it has low toxicity, high biodegradability and compatibility with human skin. This book, therefore, shows the production of certain type of rhamnolipids from Pseudomonas aeruginosa TMN which is new strain isolated from Compost pile waste collected from El-Sharkia region in Delta, Egypt. Different carbon and nitrogen sources are used for rhamnolipid production but the most favorable were glycerol and sodium nitrate. Many techniques were used for identification of produced rhamnolipid such as Fourier transform infrared spectroscopy, Nuclear magnetic resonance and Electro-spray ionization-mass spectrometry. Produced rhamnolipid have the ability to reduce surfacetension of water until 34 mN/m and have high emulsification power.