Mineral weathering plays an important role in poor-nutrient environments such as mine spoils and tailings. Ectomycorrhizal (ECM) fungi are able to enhance mineral weathering through different mechanisms, thereby increasing the availability of minerals and nutrients to plants. Six ECM fungi (Cadophora finlandia, Cenococcum geophilum, Hebeloma crustuliniforme, Lactarius aurantiosordidus, Paxillus involutes, and Tricholoma scalpturatum) were tested here for their tolerance to biotite-quartz-rich mine tailings. Either solid- or liquid-medium methods were used for in vitro selection of ECM fungi for their ability to grow on mine tailings. ECM fungi were selected based on their mycelial radial growth and metabolite production (ergosterol and low-molecular-mass organic acids, LMMOAs). We found a strong correlation between fungal ergosterol content and mycelial radial growth using the solid-medium method. However, the liquid-medium method was more appropriate for ergosterol synthesis and permitted direct measurement of organic acid production. We found that LMMOAs were exuded by ECM fungi, which solubilized mine tailings for their own growth and nutrition. Finally, we concluded that the ECM fungi C. finlandia and T. scalpturatum are the species most tolerant to tailings and could potentially improve the survival rate, growth, and health of white spruce seedlings planted on biotite mine spoils and tailings.