Rhamnolipids are bacterial biosurfactants produced by Pseudomonas spp. These compounds have been shown to present several interesting biological activities, restricting the growth of Bacillus subtilis and showing zoosporicidal activity on zoosporic phytopathogens. It has been suggested that the interaction with the membrane could ultimately be responsible for these actions. Therefore, it is of great interest to gain insight into the molecular mechanism of the interaction of purified rhamnolipids with the various phospholipid components of biological membranes. In this work, the critical micelle concentration (cmc) of a purified dirhamnolipid produced by Pseudomonas aeruginosa has been determined both by isothermal titration calorimetry and surface tension measurements. The partition coefficients from water to membranes of different compositions, as well as the corresponding thermodynamic parameters, have been determined by isothermal titration calorimetry measurements. The results indicate that dirhamnolipid membrane partitioning is an entropically driven process. The presence of cholesterol in the membrane decreases the partition of dirhamnolipid. On the other hand, phosphatidylethanolamine stimulates dirhamnolipid binding, whereas lysophosphatidylcholine opposes binding, suggesting that the biosurfactant behaves as an inverted-cone-shaped molecule. The values obtained for the cmc and the partition constant are considered in relation to the surfactant potency of dirhamnolipids.