In this study, the biosurfactant produced by Pseudomonas aeruginosa was evaluated in view of its ability to be used in Microbial-Enhanced Oil Recovery (MEOR). This microorganism was isolated from a soil artificially contaminated with crude oil and used to produce rhamnolipid using glycerol as the carbon source. The biosurfactant efficiently reduced water surface tension from 72 to 35.26 mN/m at its critical micelle concentration of 127 mg/L and emulsification rate (E 24) of 69% for the crude oil. Furthermore, it was demonstrated that the rhamnolipid can recover oil, even 2 months after its production, which shows that its biodegradability is not a disadvantage to the application in MEOR. The best result, for a biosurfactant concentration of 100% above the Critical Micelle Concentration (CMC) and petroleum with API gravity of 21.90, showed that the total recovery factor was 50.45 ± 0.79%, of which 11.91 ± 0.39% corresponds to MEOR.