Acid soils are widespread in tropical and subtropical areas in the world and contain a high concentration of active manganese (Mn) at pH lower than 5.5. Ectomycorrhizal (ECM) fungi are important for artificial forestation in acid soils and ecological restoration in Mn-polluted regions. A better understanding of resistance mechanisms to Mn and selection of proper ECM fungi are vital prerequisites for producing ECM seedlings in nursery beds. Therefore, Russula delica Fr. (Rd Fr), Pisolithus tinctorius 715 (Pt 715), Cenococcum geophilum Fr. (Cg Fr), and Suillus grevillei (Kl.) Sing (Sg KlS) were grown in liquid culture media with various Mn²⁺ concentrations, in order to study the growth, nutrient uptake (nitrogen, phosphorus, and potassium), organic acid efflux, and Mn distribution in fungal hyphae. The results showed no significant influence of Mn²⁺ on the growth of Rd Fr at concentrations between 0 and 800 mg Mn²⁺/L. Low Mn²⁺ concentrations simulated the growth of Sg KlS and no growth inhibition was observed at high Mn²⁺ concentrations. In contrast, the growth rate of Pt 715 and Cg Fr was significantly reduced by Mn²⁺, and the biomass was decreased by 22.75% for Pt 715 and 33.33% for Cg Fr at high Mn²⁺ concentrations, compared to the control (0 mg Mn²⁺/L). These results suggested that Rd Fr and Sg KlS were highly resistant to Mn²⁺, probably because of their environmental adaptation and biological evolution, as a result of their long-term habitation in acid soils with high concentrations of active Mn²⁺. A significant decrease was observed in both the content and absorption of nitrogen and potassium by ECM fungi in liquid media with Mn²⁺ added, which could be unbeneficial to host nutrient uptake after the formation of ectomycorrhizas under Mn stress. The content and uptake of phosphorus and efflux of oxalate and citrate by ECM fungi showed diverse changes among fungi grown in culture media with added Mn²⁺. These results indicated that Mn²⁺ fixation by phosphate or polyphosphates and Mn²⁺ complexation by organic acids in the process of alleviating Mn²⁺ toxicity varied among ECM fungal strains. Under Mn²⁺ stress, proton efflux rate, and Mn content in hyphae, particularly in protoplasm, of Rd Fr and Sg KlS were consistently lower than those of Cg Fr and Pt 715, the sensitive fungi to Mn²⁺. The decreased availability and absorption of Mn²⁺ could thus be considered as an important mechanism of ECM fungi to alleviate Mn²⁺ toxicity. In addition, most of Mn²⁺ absorbed by fungal hyphae located in apoplasts and only few entered into protoplasm. Mn²⁺ in apoplast ranged between 5.23 and 9.21 folds of protoplasm. Therefore, plasma membrane could be considered as an important barrier of ECM fungi against Mn²⁺ entry into the cells. Since lab culture and field conditions are different, further studies are necessary on the influence of Mn stress on tree roots and ECM fungi in the field.