The pollution and toxicological effects of microplastics in soil have gradually attracted widespread attention, but studies on the ecological effects of microplastics on soil microorganisms and their mechanisms are still very scarce. To explore the physiological response of ectomycorrhizal fungi (Lactarius delicious, Ld) to microplastic stress, Ld was used as the test strain, and 80 nm and 4 μm monodisperse polystyrene microspheres (PS-MPs) were selected. The solid plate method and the liquid culture method were used to study the effect of PS-MPs with two particle sizes on the growth, nutrient content, reactive oxygen species (ROS) production, organic acid secretion and other indicators of the strain Ld. The results showed that with increasing PS-MPs concentration, the biomass of strain Ld decreased gradually. When the strain Ld was exposed to high concentrations of PS-MPs (200-300 mg L-1), PS-MPs with particle size of 4 μm showed stronger inhibition of biomass than PS-MPs with particle size of 80 nm. After exposure to the PS-MPs environment of two sizes, the concentration of PS-MPs increased (0-300 mg L-1), and the contents of phosphorus (P), potassium (K), H2O2 and superoxide anion radical (O2·-) in Ld mycelia increased significantly (p < 0.05). This showed that the antioxidant substances in Ld could not remove excess ROS, but could reduce the stress of microplastics by increasing the absorption of P and K from the environment, which was beneficial to its own metabolic activities. In addition, the strain Ld could secrete oxalic acid, acetic acid and succinic acid into the culture medium, which the amount of oxalic acid secreted was the highest. We speculated that strain Ld responded to the stress of microplastics by secreting a large amount of organic acids. Therefore, the mechanism of the influence of microplastics on ectomycorrhizal fungi may involve oxidative stress, and the mycelia responded to the toxicity of microplastics by secreting more organic acids and increasing the absorption of P and K. The results of this study will provide a scientific basis for the study of the acute toxicity of microplastics to ectomycorrhizal fungi.