Previous studies indicated that ectomycorrhizal (EM) hyphae can access non-host plant roots and inhibit their growth, with the underlying mechanisms remaining largely unexplored. This study established a tripartite co-culture system consisting of EM fungi supported Quercus mongolica and non-host plants Arabidopsis thaliana or Setaria italica. Plant growth, nutrient concentrations, transcriptome (mRNAs and miRNAs), microbial communities, and fatty acids were determined to comprehensively understand the effects of EM on non-host plants. The results showed that roots of non-host plants were colonized by EM hyphae of Scleroderma. EM hyphae colonization significantly inhibited non-host plant growth and decreased their leaf [N], but increased leaf [P] and leaf free fatty acid concentrations. A small amount of ¹⁵N was transferred from non-host to Q. mongolica leaves. Foliar N application alleviated EM hyphae inhibited non-host plant growth. EM hyphae colonization activated expression of genes associated with plant-pathogen interaction responses but suppressed those involved in photosynthesis in A. thaliana leaves. Our findings suggest that a growth-defense trade-off, in conjunction with a leaf N: P stoichiometric imbalance, explain the observed inhibition of non-host plant growth by EM hyphae. This study provides insights into ectomycorrhiza mediated host and non-host plants interaction, which is important for plant community establishment.