This work focuses on the use of a co-product of biodiesel production as carbon and energy source for the production of rhamnolipids by a new strain of Pseudomonas aeruginosa MSIC02. Different carbon sources were compared and the highest rhamnolipid concentration (1269.79mg/L) was achieved when hydrolyzed glycerin was used. The study of nutritional and environmental conditions allowed an increase in the production of rhamnolipids. Maximum rhamnolipid concentration was attained at 18g/L of glycerol, 4.0g/L of NaNO3, 62mM of KH2PO4, pH 7.0 and 37°C. Highest product yields on substrate and biomass and productivity were achieved at the same conditions. The biosurfactant showed good emulsifying properties (IE24=65%), being able to form emulsions with mineral and vegetable oils. The NMR spectrum of H1 and C13 and mass spectra indicates that the purified product contained two types of rhamnolipids: l-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate (RL1) and l-rhamnosyl l-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate (RL2).