Pseudomonas aeruginosa produce biosurfactant with biotechnological importance through fermentation. The main factors affecting biosurfactant production are the type of organism used and the components of the fermentation medium (pH, temperature, oxygen, carbon and nitrogen sources, various salts). Increasing the production of rhamnolipid produced in low amounts has been the subject of many studies. In this study, toluene was used to increase rhamnolipid production. The addition of 0.2% toluene at the 48th h resulted in the highest rhamnolipid formation (3.0 g/L), which is a significant 30% increase over the control (2.3 g/L). While rham-nolipid production increased with the addition of toluene, bacterial biomass decreased. This study revealed that adding toluene to the fermentation medium with a new strategy significantly increases rhamnolipid production. Addition of toluene is an easy and effective way to increase rhamnolipid production in P. aeruginosa fermentation processes. The present research is the first to demonstrate that P. aeruginosa improves rhamnolipid synthesis when toluene is added.