The permeability of Norway spruce wood can be reduced to 1-5% of that of green timber when dried, resulting in only slight radial penetration of chemical solutions. We investigated the penetration of the biocide 3-iodo-2-propynyl butylcarbamate, (IPBC) in Norway spruce and Scots pine sapwood when applied in a water-based coating system. Penetration of both untreated wood and specimens pretreated with the white-rot fungus Physisporinus vitreus was analyzed by the absence or presence of blue staining after incubation with Aureobasidium pullulans and Sclerophoma pithyophila. The qualitative results were compared with quantitative data obtained by chemical analysis. It was assumed a pre-treatment with Physisporinus vitreus could make the surface of the substrate more porous and improve the penetration of fluid substances. All analytical investigations of the samples were done after an outdoor weathering period of 6 months. From a practical point of view the performance of the coated substrate is more relevant after a certain aging procedure then right after the application. The chemical measured uptake of IPBC for Scots pine was similar for all samples at the surface. The penetration depth was higher for those samples with a pre-treatment with Physisporinus vitreus. The colonization pattern of the samples by blue-stain fungi depended upon the pretreatment but did not show a clear correlation between IPBC-content and staining. The cross-section of the samples with pre-treatment was completely stained, the cross-section of the samples without pre-treatment did show a distinctive blue stain-free zone. In Norway spruce, the chemical measured uptake of IPBC was similar for all samples at the surface independent of the pre-treatment. The penetration depth was higher for those samples with a pre-treatment with Physisporinus vitreus. But a clear visible colonization by blue-stain fungi was only observed for the samples with a pretreatment with Physisporinus vitreus. The samples without pre-treatment were locally stained across the entire cross-section. As with the Scots pine, a clear correlation between IPBC-content and blue staining was not visible, following comparison of the samples with and without pre-treatment with Physisporinus vitreus. The results suggest that not only the presence of IPBC influences colonization by A. pullulans and S. pithyophila, but also a range of other factors in wood pre-treated with P. vitreus.