Surface tension related properties of pure rhamnolipids R1 (Rha-C-10-C-10), R2 (Rha-Rha-C-10-C-10) and their mixtures with R2/R1 molar ratios of 0.69, 1.07, and 1.2 are investigated at their natural pH with and without bulk phase additives. Surface tension, dynamic light scattering, UV-vis spectrophotometric measurements and observations by scanning electron microscope and cross polarized light microscope were used to evaluate the surface adsorption and spontaneous aggregation of rhamnolipid mixtures in aqueous solutions. Structural changes taking place in the surface monolayers with an increase in the bulk concentration of rhamnolipid mixtures were evaluated through analysis of surface tension measurements. Interactions of the two hydrophilic groups, carboxylic acid and rhamnosyl groups, of the rhamnolipids through electrostatic forces and hydrogen bonds, respectively, were elucidated with the use of electrolytes and rhamnose in the bulk phase. Self-assembly of rhamnolipids correlated well with the state of the monolayer. Vesicle formation was observed at concentrations below the critical micelle concentration (cmc). This critical concentration,for spontaneous vesicle formation was correlated with the onset of condensed phases in the monolayer. Micelles could only be observed at very high concentrations, about hundred times the cmc, in solutions of R2 and its mixtures with R1. Vesicles of R2 were found to be more uniform than the vesicles of R1. This difference in aggregation behavior could be due to the different stabilization mechanisms of the aggregates. (C) 2016 Elsevier B.V. All rights reserved.