Copper-based formulations of wood preservatives are widely used in industry to protect wood materials from degradation caused by fungi. Wood treated with preservatives generate toxic waste that currently cannot be properly recycled. Despite copper being very efficient as an antifungal agent against most fungi, some species are able to cope with these high metal concentrations. This is the case for the brown-rot fungus Rhodonia placenta and the white-rot fungus Phanerochaete chrysosporium, which are able to grow efficiently in pine wood treated with Tanalith E3474. Here, we aimed to test the abilities of the two fungi to cope with copper in this toxic environment and to decontaminate Tanalith E-treated wood. A microcosm allowing the growth of the fungi on industrially treated pine wood was designed, and the distribution of copper between mycelium and wood was analysed within the embedded hyphae and wood particles using coupled X-ray fluorescence spectroscopy and Scanning Electron Microscopy (SEM)/Electron Dispersive Spectroscopy (EDS). The results demonstrate the copper biosorption capacities of P. chrysosporium and the production of copper-oxalate crystals by R. placenta. These data coupled to genomic analysis suggest the involvement of additional mechanisms for copper tolerance in these rot fungi that are likely related to copper transport (import, export, or vacuolar sequestration).