Wool fibres are subject to a variety of industrial chemical treatments prior to finishing processes. The influence of such treatments on subsequent enzymatic treatments is still poorly characterised, but its importance is paramount to achieve maximal efficiency of enzymatic action on the fibres. The effect of such treatments on wool fibre structure and their significance on the accessibility of the enzymes transglutaminase and tyrosinase to their target amino acids within wool proteins was investigated. These enzymes are of great interest due to their potential as protein cross-linkers and their capability to covalently graft beneficial compounds onto proteins. The accessibility of target amino acids was assessed using specific peptides tagged with fluorescent probes, followed by epifluorescence or confocal microscopy. Differences in the action of the different chemical pretreatments were found, with a treatment with permonosulphuric acid resulting in the greatest level accessibility to both enzymes, followed by chlorination and plasma treatments. This was confirmed by the increase in tensile strength of treated wool observed in the enzyme-treated yarns. This work illustrates that the accessibility of these enzymes to their target aminoacids in solid substrates such as wool is key for their action and to achieve the desired property modifications. The techniques used have the advantage of permitting the visual monitoring of the diffusion of the active enzymes through the wool fibre structure and to relate this to the extent of enzymatic reaction. Crucially, this will allow an informed choice of the best wool processing stage at which to introduce such enzyme treatments.