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A current challenge in nanomedicine is to develop innovative strategies to fight infections caused bymultiresistant bacterial pathogens. A striking example is antiadhesion therapy, which represents an attractive alternative to antibiotics to prevent and treat biofilm-associated infections on medical devices. By means of single-cell force nanoscopy, we demonstrate that sophorolipid (SL) biosurfactants feature unusually strong antiadhesion properties against Staphylococcus aureus and Escherichia coli, two nosocomial pathogens involved in catheter-related infections, which represent a major public health problemworldwide.We find that the nanoscale adhesion forces of single bacteria are much weaker on SL monolayers than on abiotic alkanethiol monolayers. The remarkable antifouling efficacy of SL-surfaces is likely to involve repulsive hydration forces associated with sophorose headgroups. We also show that, owing to their surfactant properties, soluble SLs block bacterial adhesion forces towards abiotic surfaces. Collectively, our single-cell experiments demonstrate that sophorolipids exhibit strong and versatile antiadhesion properties, making them promising candidates to design anti-infective biomaterials.