- We investigated the influence of feed-water shear rate during reverse-osmosis (RO) desalination on biofouling with respect to microbial community composition developed on the membrane surface. The RO membrane biofilm's microbial community profile was elucidated during desalination of tertiary wastewater effluent in a flat-sheet lab-scale system operated under high (555.6 s−1), medium (370.4 s−1), or low (185.2 s−1) shear rates, corresponding to average velocities of 27.8, 18.5, and 9.3 cm s−1, respectively. Bacterial diversity was highest when medium shear was applied (Shannon–Weaver diversity index H' = 4.30 ± 0.04) compared to RO-membrane biofilm developed under lower and higher shear rates (H′ = 3.80 ± 0.26 and H′ = 3.42 ± 0.38, respectively). At the medium shear rate, RO-membrane biofilms were dominated by Betaproteobacteria, whereas under lower and higher shear rates, the biofilms were dominated by Alpha- and Gamma- Proteobacteria, and the latter biofilms also contained Deltaproteobacteria. Bacterial abundance on the RO membrane was higher at low and medium shear rates compared to the high shear rate: 8.97 × 108 ± 1.03 × 103, 4.70 × 108 ± 1.70 × 103 and 5.72 × 106 ± 2.09 × 103 copy number per cm2, respectively. Interestingly, at the high shear rate, the RO-membrane biofilm's bacterial community consisted mainly of populations known to excrete high amounts of extracellular polymeric substances. Our results suggest that the RO-membrane biofilm's community composition, structure and abundance differ in accordance with applied shear rate. These results shed new light on the biofouling phenomenon and are important for further development of antibiofouling strategies for RO membranes.