Microclimatic conditions in forests depend on canopy cover and thus differ between closed and open forests and similarly change along the vertical axis from the forest floor to the upper canopy. Yet, it is unknown whether this similarity in microclimatic conditions in forest gaps and the upper canopy provides equivalent habitats for wood-inhabiting species assemblages and thus, whether deadwood in the canopy is needed to maintain biodiversity. We tested this question for wood-inhabiting beetles, fungi and bacteria, using 150 branch bundles of Picea abies, Abies alba and Fagus sylvatica experimentally exposed along a vertical and horizontal gradient in microclimate: sun-exposed in the upper canopy and in gaps, half-shaded in the mid canopy and at the edge of forest gaps and shaded near ground under a closed canopy in a temperate forest in Central Europe. Variation partitioning showed that assemblage composition of all taxa was predominantly determined by tree species, followed by vertical stratum in beetles. Beetle assemblages at upper and mid-canopy formed a distinct community compared to those near ground. Fungal assemblages in the upper canopy were similar to those in gaps near the ground, while bacteria assemblages in the upper canopy were different only from those near ground under closed canopy. Our results indicate that the canopy stratum and sun-exposed deadwood enlarge the niche space for saproxylic organisms. Conservation strategies should thus aim at retaining habitat trees of different tree species with deadwood in the canopy as well as gaps with sun-exposed deadwood, e.g., after disturbances, to maintain biodiversity.