Rhamnolipids are bacterial biosurfactants produced by Pseudomonas spp. These compounds have been shown to present several interesting biological activities and to have potential applications as therapeutics agents. It has been suggested that the interaction with the membrane could be the ultimate responsible for these actions. Therefore it is of great interest to get insight into the molecular mechanism of the interaction of purified rhamnolipids with the various phospholipid components of biological membranes. In this work, the CMC of a purified bacterial dirhamnolipid was 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, indicated that membrane partitioning was an entropically driven process. Interaction of dirhamnolipid with phospholipids was studied by means of calorimetry, FTIR and X-ray diffraction. It is shown this interaction had various effects that might constitute the molecular basis to explain the former activities: domain formation with lateral phase separation, increased motional disorder of the phospholipid acyl chains and dehydration of the aqueous interface. Our results suggest that dirhamnolipid, having a large polar headgroup and a smaller hydrophobic portion, behaves as an inverted-cone shaped molecule, conferring positive curvature to membranes, which might be behind its disrupting effects on membranes.