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Large-scale replacements of native birch with spruce have been carried out in Western Norway for economic reasons. This tree species shift potentially affects biotic components such as the eucaryome, consisting of microscopic animals (Metazoa), protists and fungi, which are key players in the functioning of forest ecosystem. The impact on the belowground eukaryome and its interactions with vegetation and soil properties is not well assessed. We examined the impact of replacing native birch with Norway spruce plantations on the eukaryome of the boreal forest floor in Western Norway using 18S rDNA metabarcoding. The tree species shift from birch to spruce had significant impacts on the eukaryome at both taxonomic (Metazoa) and functional categories (phagotrophs, phototrophs, parasites and osmotrophs). The distinct differences in eukaryome communities were related to changes in understorey vegetation biomass and soil chemistry following the tree species shift. This had a negative effect on eukaryome richness, particularly affecting phagotrophs and parasites, while the opposite was observed for osmotroph richness. Our results indicated that the spruce plantations altered the eukaryome communities and their food-web patterns compared to what was found in the native birch forest soil. This information should be taken into consideration in forest management planning.