Coniferous trees can take up mineral nutrients either non-symbiotically from nutrient-rich hotspots in the forest floor or via symbiosis with ectomycorrhizal (EcM) fungi; EcM symbiosis also enables the trees to obtain nutrients from organic substances. Excellent examples of nutrient-rich forest-floor hotspots are abandoned wood ant nests, which accumulate significant amounts of inorganic substances that are readily accessible to roots as well as organic substances that are not accessible to non-mycorrhizal roots. We examined the effects of substrates from abandoned nests of the wood ant Formica polyctena and EcM symbiosis on the growth of Norway spruce (Picea abies) seedlings. The tested substrates originated from the centers and rims of wet and dry ant nests, and from the surrounding forest floor. Aseptic spruce seedlings were grown in these substrates in a growth chamber, and after 7 months, seedling biomass, the chemical and microbiological properties of the substrates, and EcM colonization and diversity were determined. Spruce seedlings grew better and had a more diverse spectra of EcM fungi in the ant nest substrates than in the forest floor substrate. Substrate nutrient content, especially phosphorus and basic cations, positively correlated with spruce biomass, EcM diversity, and fungal biomass. Contrary to conventional wisdom, high nutrient contents in ant nests apparently enhanced EcM abundance and diversity. Although abandoned wood ant nests in temperate and boreal forests are not abundant and thus are unlikely to have ecosystem level effects, they may cause significant local variations in tree growth and in the occurrence of root-symbiotic fungi.