To prevent indoor fungal growth, understanding the moisture relations of fungi is a key element. Indoor moisture is quantified by the relative humidity (RH).RHcontrols the water activity of the indoor materials that fungi grow on, a well-studied parameter known to limit fungal growth.RH, however, also controls the amount of water present in these materials, the moisture content. The significance of the moisture content of these materials to indoor fungal growth is currently overlooked. In the work reported here, growth experiments with the indoor fungusPenicillium rubenson gypsum substrates were performed to test whether the moisture content influences growth on porous materials. Second, we report the development of a video microscopy method that for the first time quantified hyphal growth on a porous material. It is found that a higher moisture content leads to earlier colonization and higher hyphal extension rates. This is a fundamental step in unravelling the effect ofRHon indoor fungal growth. The real-time monitoring of colonization of gypsum provides a new view of growth on indoor surfaces.