Due to the increasingly stringent energy efficiency requirements, timber frame houses are becoming more and more popular across Europe. Depending on the circumstances, timber frame walls might be exposed to high relative humidity or condensation conditions, leading to a risk of fungal degradation. In order to assess the durability of a timber frame construction in a reliable way, a clear view on the potential risk of fungal growth is essential. Various experiments are reported in literature to define the minimal moisture threshold for fungal decay, yet all very different in set-up and hence leading to contradictory conclusions. Therefore, the present paper aims at a contribution to a better understanding of the influence of moisture conditions on the onset and progress of wood decay in timber frame walls. Based on the information that is still lacking in international literature, a new test methodology has been elaborated within this work and is reported in this paper.