Rhamnolipids are very promising sugar-based biosurfactants, generally produced by bacteria, with a wide range of properties that can be exploited at an industrial and technological level, e.g. in cosmetics, food science, or oil recovery, to provide benefits for human health and the environment. This has led to intensive research into optimizing their production to increase yields and minimize costs, which is challenging because biotechnological methods for rhamnolipid production result in complex product mixtures and require the introduction of complex separation strategies to ensure the purity of the rhamnolipid obtained. This is an important issue for the introduction of rhamnolipids to the market due to the differences that exist between the properties of the different congeners. This review attempts to provide an overview of the interfacial properties, potential applications, and recent advances in understanding the molecular mechanisms that govern the adsorption to interfaces and assembly in solution of rhamnolipids. In addition, the review also discusses some general aspects related to the production and purification methods of rhamnolipids, highlighting the need for further research to fully exploit their potential. It is hoped that this review will contribute to the growing body of knowledge about rhamnolipids and stimulate further research in this field.