In the present study, we investigate the possible contribution of Sphingomonas spp. glycosphingolipids (GSL) and its extracellular polymeric substances (EPS) to the initial colonization and development of biofilm bodies on reverse osmosis (RO) membranes. A combination of an RO cross-flow membrane lab unit, a quartz crystal microbalance with dissipation (QCM-D), and a rear stagnation point flow (RSPF) system with either model bacteria (Sphingomonas wittichii, Escherichia coli, and Pseudomonas aeruginosa) or vesicles made of the bacterial GSL or LPS was used. Results showed noticeable differences in the adhesion LPS versus GSL vesicles in the QCM-D, with the latter exhibiting 50% higher adhesion to polyamide coated crystals (mimicking an RO membrane surface). A similar trend was observed for EPS extracted from S. wittichii, when compared to the adhesion tendency of EPS extracted from P. aeruginosa. By applying the whole-cell approach in the RO lab unit, the cumulative impact of S. wittichii cells composing GSL and probably their EPS reduced the permeate flux during bacterial accumulation on the membrane surface. Experiments were conducted with the same amount of Sphingomonas spp. or Escherichia coli cells resulting in a two times greater flux decline in the presence of S. wittichii. The distinct effects of Sphingomonas spp. on RO membrane biofouling are likely a combination of GSL presence (known for enhancing adhesion when compared to non-GSL containing bacteria) and the EPS contributing to the overall strength of the biofilm matrix.