Various studies have described remarkable biological activities and surface-active properties of rhamnolipids, leading to their proposed use in a wide range of industrial applications. Here, we report on a study of the effects of monorhamnolipid RhaC10C10 and dirhamnolipid RhaRhaC10C10 incorporation into model membranes of varying complexity, including bacterial and heterogeneous model biomembranes. For comparison, we studied the effect of HAA (C10C10, lacking a sugar headgroup) partitioning into these membrane systems. AFM, confocal fluorescence microscopy, DSC, and Laurdan fluorescence spectroscopy were employed to yield insights into the rhamnolipid-induced morphological changes of lipid vesicles as well as modifications of the lipid order and lateral membrane organization of the model biomembranes upon partitioning of the different rhamnolipids. The partitioning of the three rhamnolipids into phospholipid bilayers changes the phase behavior, fluidity, lateral lipid organization and morphology of the phospholipid membranes dramatically, to what extent, depends on the headgroup structure of the rhamnolipid, which affects its packing and hydrogen bonding capacity. The incorporation into giant unilamellar vesicles (GUVs) of a heterogeneous anionic raft membrane system revealed budding of domains and fission of daughter vesicles and small aggregates for all three rhamnolipids, with major destabilization of the lipid vesicles upon insertion of RhaC10C10, and also formation of huge GUVs upon the incorporation of RhaRhaC10C10. Finally, we discuss the results with regard to the role these biosurfactants play in biology and their possible impact on applications, ranging from agricultural to pharmaceutical industries.