Acetylation is a commercialised chemical wood modification technology that increases the durability of wood against microbial attack. However, the details of how acetylation protects the wood structure from fungal degradation are still unclear. In this study, we tested the hypothesis that the resistance against microbial attack depends on the localisation of acetylation within the cell wall. The methodology involved two types of acetylation (uniform and lumen interface modification), which were analysed by lab-scale degradation with Rhodonia placenta, chitin quantification, infrared spectroscopy, and Raman microspectroscopy. The location of the acetylation did not affect overall mass loss during degradation experiments. Instead, the mass loss was related to the intensity of the treatment. However, chemical imaging of the interface acetylated specimens showed that degradation primarily took place in cell wall regions that were less acetylated. It was also observed that the fungus required more fungal biomass (i.e., fungal mycelia) to degrade acetylated wood than untreated wood. Based on dimensions and comparison to a reference spectrum, several cross-sections of hyphae located within lumina were discovered in the Raman images. These hyphae showed presence of chitin, water and chelated metals within their walls, and could be separated into an inner and an outer part based on their chemistry as seen in the spectra. The outer part was distinguished by a relatively higher amount of water and less chelated iron than the inner part.