The timberline ecotone is a sensitive area to climate change among the terrestrial ecosystems, and it is vital to study the soil fungal community structure and its formation mechanism in the timberline ecotone to predict the effect of climate change on soil nutrient cycles and to maintain terrestrial ecosystem functions. For this, we collected soil samples from the shrub and coniferous forests of Shennongjia National Park. The soil fungal community structure and diversity were analyzed using the Illumina sequencing method. The results showed that the dominant soil fungal phyla, genera, and species differed between the two forest types. Basidiomycota was the dominant phylum in the coniferous forest, whereas Zygomycota was dominant in the shrub forest. Detrended correspondence analysis and dissimilarity test indicated that their soil fungal communities were significantly different, and the soil fungal diversity was significantly higher (P<0.05) in the coniferous forest than in the shrub forest according to the Shannon index, Chao value, and richness index. Canonical correlation analysis and Mantel test indicated that the soil pH, plant diversity, soil moisture, and temperature had significant linkages with the soil fungal community structure. Therefore, the soil fungal community structure and diversity in the timberline ecotone were significantly different, and the factors of soil pH,plant diversity, soil moisture, and temperature were the important environmental factors that shaped the fungal community structure.