The preparation layers (gesso) applied on archaeological wood have a slightly degrading effect on the wood surface that lies directly beneath them. This research seeks to investigate the effect of preparation layers according to their penetration depth within the wood using SEM-EDX. Native Ficus sycomorus (sycomore fig), Tamarix sp. (tamarisk wood) and Acacia sp. (syn. Vachellia sp., acacia wood) and six types of preparation layers, similar in composition to ancient paint grounds were chosen for this study. For investigation and analysis, samples were taken from the wood at a depth of 5 mm directly below the substrate, after exposing the samples to natural and accelerated (light and heat) aging, which simulated partially the natural ageing process of ancient Egyptian artifacts. Results of investigation and analysis were compared with results obtained from the control wood samples that had not been covered with any preparation layers. The results concluded that penetration of preparation layers inside the wood structure is totally dependent on three factors; first, the wood type which differed in pore size, second, the mineralogical composition of the preparation layer applied on the wood, and third the binding medium used.