- Bacterial outer membrane components play a critical role in bacteria–surface interactions (adhesion and repulsion). Sphingomonas species (spp.) differ from other Gram-negative bacteria in that they lack lipopolysaccharides (LPSs) in their outer membrane. Instead, Sphingomonas spp. outer membrane consists of glycosphingolipids (GSLs). To delineate the properties of the outer membrane of Sphingomonas spp. and to explain the adhesion of these cells to surfaces, we employed a single-component-based approach of comparing GSL vesicles to LPS vesicles. This is the first study to report the formation of vesicles containing 100% GSL. Significant physicochemical differences between GSL and LPS vesicles are reported. Composition-dependent vesicle adherence to different surfaces using quartz crystal microbalance with dissipation monitoring (QCM-D) technology was observed, where higher GSL content resulted in higher mass accumulation on the sensor. Additionally, the presence of 10% GSL and above was found to promote the relative rigidity of the vesicle obtaining viscoelastic ratio of 30–70% higher than that of pure LPS vesicles.