Bitelechelic polydimethylsiloxanes (PDMS) and 2-hydroxyethylacrylate (HEA)/acrylic acid(AA) were photopolymerized to give nanophase separated amphiphilic 20-mum-thin coatings covalently attached to glass. The coatings quickly take up the antimicrobial surfactant cetyltrimethylammonium chloride (CTAC). After a 30 min loading period the release of CTAC in water was followed by simultaneously measuring both the antimicrobial activity of the coating's surface against Staphylococcus aureus and the release of the structurally related dye Rhodamine B. Depending on the composition the antimicrobial activity remained up to 3 weeks. The influence of different pH, varying sodium chloride concentrations, and the surfactant Pluronic was investigated. However, even in the cases of 160 mM NaCl and 1 wt.% Pluronic as release medium, the antimicrobial activity remained more than 6 days in case of a coating consisting of 45 wt.% PDMS, 31 wt.% PHEA, and 25 wt.% PAA. Mechanistic investigations revealed that the CTAC-loaded coatings act like contact-active surfaces, i.e., they do not kill microbes in the surrounding solution but only on their surface. This supports our hypothesis that the antimicrobial action is due to a concentration gradient of CTAC on the surface, allowing it to be antimicrobial on contact and to release only very low concentration of the biocide into the surrounding.