AimsThis study investigates pinewood nematode's impacts on root mycoflora of Pinus tabulaeformis. Methods and ResultsThe biomass, colonization rate, community structure and diversity of root-associated fungi were investigated in pinewood nematode-infected and nematode-noninfected P. tabulaeformis. The results indicated that the roots of P. tabulaeformis were colonized highly by root-associated fungi, mainly ectomycorrhizal fungi (ECMF) and dark septate endophytes. Infection of pinewood nematode was associated with a significant (P<005) decrease in root colonization rates by ECMF, dark septate endophytes and total hyphae, as well as in fungal biomass in the roots. Illumina MiSeq sequences of tagged amplicons of 18S rDNA region revealed Basidiomycota (6570%) and Ascomycota (3414%) as the dominant root-associated fungi in roots of P. tabulaeformis. Among the detected operational taxonomic units (OTUs), ECMF and dark septate endophytes exhibited a higher relative abundance in trees infected by pinewood nematode compared with noninfected ones. ConclusionsThe infection of pinewood nematode altered the composition and OTU abundance of root-associated fungi community in P. tabulaeformis roots with a decrease in the biomass, species richness and diversity of root-associated fungi, as well as in the colonization rates and abundance of ECMF and dark septate endophytes. Significance and Impact of the StudyThis study is an important contribution for better understanding the interaction between pine wilt disease and root-associated fungi.