Biological amphiphiles derived from natural resources are presently being investigated in the hope that they will someday replace current synthetic surfactants, which are known pollutants of soils and water resources. Sophorolipids constitute one of the main classes of glycosylated biosurfactants that have attracted interest because they are synthesized by non-pathogenic yeasts from glucose and vegetable oils at high titers. In this work, the self-assembly properties of several sophorolipids in water at high concentrations (20–80 wt %), a range so far mostly uncharted, have been investigated by polarized-light microscopy and X-ray scattering. Some of these compounds were found to show lyotropic liquid-crystalline behavior as they display lamellar or hexagonal columnar mesophases. X-ray scattering data shows that the structure of the lamellar phase is almost fully interdigitated, which is likely due to the packing difference between the bulky hydrophilic tails and the more compact aliphatic chains. A tentative representation of the molecular organization of the columnar phase is also given. Moreover, some of these compounds display thermotropic liquid-crystalline behavior, either pure or in aqueous mixtures. In addition, small domains of the lamellar phase can easily be aligned by applying onto them a moderate a.c. electric field, which is a rather unusual feature for lyotropic liquid crystals. Altogether, our work explored the self-assembly liquid-crystalline behavior of sophorolipids at high concentration, which could shed light on the conditions of their potential industrial applications as well as on their biological function.