Nanoemulsions are used in the food, cosmetics, personal care and pharmaceutical industries to provide desirable optical, textural, stability, and delivery characteristics. In many industrial applications, it is desirable to formulate nanoemulsions using natural ingredients so as to develop label-friendly products. Rhamnolipids are biosurfactants isolated from certain microorganisms using fermentation processes. They are glycolipids that have a polar head consisting of rhamnose units and a non-polar tail consisting of a hydrocarbon chain. In this study, the interfacial characteristics of this natural surfactant at medium chain triglyceride (MCT) oil-water interfaces were characterized, and its ability to form nanoemulsions was compared to that of another natural surfactant (quillaja saponins). The influence of rhamnolipid concentration, homogenization pressure, and oil type on the mean droplet diameter of emulsions produced by microfluidization was determined. Rhamnolipids were highly effective at forming small droplets (d(32) < 0.15 mu m) at low surfactant-to-oil ratios (SOR < 1:10) for MCT oil. Rhamnolipids could also be used to form small droplets using long chain triglyceride oils, such as corn and fish oil. Rhamnolipid-coated droplets were stable to aggregation over a range of pH values (5-9), salt concentrations (< 100 mM NaCl) and temperatures (20-90 degrees C). However, droplet aggregation was observed at highly acidic (pH 2-4) and high ionic strength (200-500 mM NaCl) conditions. These effects were attributed to a reduction in electrostatic repulsion at low pH and high salt levels. Rhamnolipid-coated droplets had a high negative charge at neutral pH that decreased in magnitude with decreasing pH. These results indicate that rhamnolipids are effective natural surfactants that may be able to replace synthetic surfactants in certain commercial applications. (C) 2015 Elsevier Inc. All rights reserved.