The development of supported glycosylated lipid layers is an important trend in the field of glyconanomaterials for their interest in understanding sugar-sugar and protein-sugar interactions, these being at the core of cellular, bacterial or viral adhesion. The conventional self-assembled monolayer (SAM) approach generally requires a thiolated glycoconjugate and a gold substrate. In this work, we show how glycolipid amphiphiles of natural origin, commonly known as microbial biosurfactants, can be easily deposited onto a substrate. Spontaneously produced by microorganisms but lacking a thiol group, one can take advantage of their self-assembly properties to prepare homogeneous supported lipid monolayers (SLM). We then choose a saturated glucolipid, G-C18:0, which forms a colloidal lamellar phase under diluted conditions. The lamellae can then be deposited onto a substrate (silicon, gold) using a physical method (dip coating). Dip coating is preferred over more classical deposition methods (Langmuir-Blodgett-LB-, vesicle fusion or spin-coating) because of its versatility, compatibility with aqueous solutions and robust control of the thickness below 10 nm. Defect-free glycosylated SLM from a microbial biosurfactant are then easily developed. A combination of ellipsometry, fluorescence microscopy, atomic force microscopy and infrared nanospectroscopy (AFMIR) show that the glycosylated SLM are defect-free, have a thickness of 2.8 ± 1.0 nm and they are highly homogeneous at scales going from the nm to cm.