Studying the fungal communities in the wood, in particular during the first events of the colonization, and the factors that underlie the dynamics of fungal species assemblages remain a challenge in ecology, because of the absence of fructification during the pioneer steps of wood degradation. The use of Next-generation DNA sequencing methods, which produce massive volumes of data, provided new perspectives in fungal molecular ecology. In this paper we used high throughput sequencing to identify factors influencing fungal colonisation during the early stages of wood decay. For this purpose wood pickets, from different wood species, were buried in an experimental site, located on the island of Oléron, and removed after six and nine months of incubation. Then, total DNA was extracted and a library of the fungal internal transcribed spacer (ITS-1) region was prepared for 454 pyrosequencing. Our results revealed a strong host effect on the fungal communities associated to different wood species together with a spatial effect on fungal diversity. The source of inoculum was also investigated with the same approach from soil samples, and results demonstrated that the major source of fungal inoculum was not located, or very weakly represented, in the close environment of our pickets. Finally, the application of high throughput sequencing approaches was investigated to improve standards in order to be more representative of natural conditions.