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Polymethylmethacrylate-based microcapsules containing the antimicrobial agent 2-n-octyl-4-isothiazolin-3-one (OIT) decorated by an anchored polyelectrolyte brush consisting of an amphiphilic diblock copolymer of polymethylmethacrylate-block-poly(sodium methacrylate) type have been formulated via a coacervation technique. The polyelectrolyte brush surface provided the microcapsule with a high and stable surface charge density. This enabled further surface modification of the colloidal particle with a thin and dense polyelectrolyte multilayer using the layer-by-layer technique. The addition of the highly charged and hydrophilic polyelectrolyte multilayer assembled on the microcapsule surface resulted in a considerable decrease of the release rate of the encapsulated OIT in aqueous suspension, corresponding to a 40 times reduction of the effective OIT diffusion coefficient in the polymethylmethacrylate matrix. Moreover, the release of encapsulated or freely dispersed OIT from coatings as a function of the matrix density was evaluated and analyzed within the framework of applied diffusion models. Encapsulation of OIT in polyelectrolyte multilayer composite microcapsules was found to significantly prolong the release and render the release rate more or less independent of the matrix density. In addition, the long-term antimicrobial properties of the coatings were evaluated in terms of their susceptibility for biofouling using the fungus and common biofouler Aspergillus niger as model organism. The results clearly demonstrated that the use of encapsulated OIT gave a significantly prolonged surface protection and allowed for the determination of the critical surface flux. The polyelectrolyte multilayer has therefore been recognized as the rate-determining barrier for OIT. The matrix density has a minor influence on the release rate of encapsulated OIT from these microcapsules and this concept may very well be expanded to cover a broad range of hydrophobic and semi-hydrophobic biocides.