Biofouling is a ubiquitous problem in many places in society and technology, especially in reverse osmosis or nanofiltration (NF) processes. Initial stages in the development of the biofilm include attachment of bacteria to the surface, where bacterial outer membrane components such as biopolymers, lipids, and proteins play important roles. Here we show that the glycosphingolipid (GSL) unique to Sphingomonas species is a key player in the initial attachment of bacteria to NF membranes whereas lipopolysaccharide (LPS), the major glycolipid in many Gram-negative species, is less significant. GSL and LPS were deposited on an NF membrane with subsequent bacterial culture injection in a three-dimensionally printed microfluidic flow cell. Flux, rejection, and pressure changes showed that GSL caused permanent study underlines the significance of Sphingomonas for the initial attachment of bacteria to membranes. and identification of key components in the biofouling process may help define strategies for biofilm A membrane fouling. This A deeper understanding prevention.