A natural glycolipid exposes the outer membrane interface as a tunable regulator of bacterial surface attachment.
Surface attachment initiates bacterial colonization and biofilm formation, yet remains difficult to target owing to redundant and species-specific mechanisms. Here we identify dalberoside, a natural glycolipid that inhibits adhesion by non-disruptive remodeling of the Gram-negative outer membrane interface. Dalberoside inhibited Pseudomonas aeruginosa and Acinetobacter baumannii attachment and biofilm formation without measurable bactericidal activity or membrane permeabilization. It reduced P. aeruginosa retention on epithelial cells, delayed P. aeruginosa cytotoxicity and rapidly detached surface-associated A. baumannii. High-resolution microscopy of a fluorescent analogue and in situ cryo-electron tomography support a model in which dalberoside associates with the bacterial outer membrane, remodeling its outer leaflet. Biophysical measurements further indicated altered local interfacial properties. Thus, dalberoside reveals the outer membrane interface as a chemically addressable regulator of bacterial surface attachment.