Sugar-based amphiphiles are a relevant natural alternative to synthetic ones due to their biodegradable properties. An understanding of their structure-assembly relationship is needed to allow the concrete synthesis of suitable derivatives. Here, four different mannose-derivative surfactants are characterized by pendant drop, dynamic light scattering, small-angle X-ray scattering, cryotransmission electron microscopy, and molecular dynamics techniques in aqueous media. Measurements denote how the polysaccharide average degree of polymerization () and the addition of a hydroxyl group to the hydrophobic tail, and thus the presence of a second hydrophilic moiety, affect their self-assembly. A variation in the of the amphiphile has no effect in the critical micelle concentration in contrast to a change in the hydrophobic molecular region. Moreover, high- amphiphiles self-assemble into spherical micelles irrespective of the hydroxyl group presence. Low- amphiphiles with only one hydrophilic moiety form cylindrical micelles, while the addition of a hydroxyl group to the tail leads to a spherical shape.