The white rot fungus Physisporinus vitreus is currently tested for several biotechnological applications such as permeability improvement of refractory wood species or the optimization of the acoustic properties of wood for violins. The enzymatic activity of P. vitreus results in the degradation of pit membranes and simultaneous alterations of the tracheid cell wall structure in wood of Norway spruce wPicea abies (L.) Karstx. By this means, selective delignification and simultaneous degradation occur in the latewood tracheids at short incubation times. To study the delignification of individual cell wall layers in latewood tracheids, cellular UVmicrospectrophotometry was applied to wood of Norway spruce that had been incubated for between 3 and 9 weeks. By means of this technique, the progressing delignification was demonstrated in the latewood tracheid secondary walls. Moreover, local delignification in close proximity to hyphal tunneling, cavities, and notches was evident. Additionally, the mechanical changes were measured (a) at the macroscopic level by Brinell hardness test and (b) at the cellular level by nanoindentation. Brinell hardness was significantly reduced with increasing incubation time which was attributed to the partial delignification. Unlike Brinell tests, results from nanoindentation tests did not show a clear effect of fungal activity because of the material heterogeneity and the high spatial resolution of this technique. The present study provides methodological approaches for the investigation of wood-fungus interactions and contributes to a better understanding of the characteristics of wood decay at the subcellular level caused by the white rot fungus P. vitreus. Moreover, it establishes the basis for a subsequent chemical analysis, for which the results will be the topic of a second paper in this series.