- Biofouling of membranes is a persistent challenge in many water purification processes including desalination. A biofilm consists of communities of organisms adhering to surfaces, thus increasing the anti-bacterial activity of the surfaces might inhibit biofilm formation and reduce the biofouling problem. Antimicrobial peptides (AMPs) attached to various surfaces for prevention of biofilm can be widely applicable for water purification technology. However, the mechanism of action of tethered AMPs is still poorly understood. Here we show a facile method to covalently attach AMPs on RO membrane surfaces, achieved by incorporation of the photoreactive 3-(4-benzoylphenyl)alanine (Bpa) into the AMP sequence RWRWRWA-(Bpa), concentration of the peptide on the surface by filtration, and subsequent irradiation with UV light. Bacteria viability on modified surfaces decreased up to 55% and biofilm inhibition was observed using Pseudomonas aeruginosa . The modification increased the surface hydrophilicity, reducing the water contact angle from 70° ± 5 to 54° ± 7, and moderately altered the performance of the membrane: permeate flux was reduced by 16–37%, and NaCl rejection increased 6%. This efficient modification method for immobilizing AMPs onto surfaces is broadly applicable to many types of membrane materials and may facilitate investigations of the mode of action of surface-tethered AMPs.