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This contribution reports an efficient method for the production and use of biocide-loaded cellulose acetate nanoparticles. As well-known model biocides 4-Hexylresorcinol and Triclosan were used for in situ nanoparticle loading during a nanoprecipitation process. We show that the nanoparticle size can be well-controlled by variation of the cellulose acetate concentration during nanoprecipitation. Apart from strong evidence suggesting cellulose acetate particle formation according to a nucleation-aggregation mechanism, we further show that the biocide loading of the particles occurs by a diffusion process and not via co-precipitation. The quantity of particle loading was analyzed by 1H-NMR spectroscopy of re-dissolved nanoparticles, and it was observed that a decisive factor for high packaging efficiency is the use of a biocide with low water solubility and high hydrophobicity. SEM studies showed no influence on the particle morphology or size by both biocides 4-Hexylresorcinol and Triclosan. Finally, an aqueous nanoparticle dispersion can be coated onto model paper sheets to yield pronounced antimicrobial surface-properties. Nanoparticles loaded with the biocide Triclosan showed a high antimicrobial activity against Bacillus subtilis, a cellulase producing bacteria, if applied to model paper substrates, even at extremely low coating weights of 1–5 g/m2, respectively. Additional long-term efficacy renders these nanoparticles ideal for various applications.