Soil moisture content (SWC) can change the diversity and composition of soil fungal communities by affecting soil texture and soil nutrients. To explore the response of soil fungal communities to moisture in the grassland ecosystem on the south shore of Hulun Lake, we set up a natural moisture gradient that was subdivided into high (HW), medium (MW), and low (LW) water contents. Vegetation was investigated by quadrat method, and aboveground biomass was collected by the mowing method. Soil physicochemical properties were obtained by internal experiments. The composition of the soil fungal community was determined using high-throughput sequencing technology. The results showed significant differences in soil texture, nutrients, and fungal species diversity under the moisture gradients. Although there was significant clustering of fungal communities in different treatments, the fungal community composition was not significantly different. According to the phylogenetic tree, the Ascomycota and Basidiomycota were the most important branches. The fungal species diversity was smaller when SWC was higher, and in this environment (HW), the fungal-dominant species were significantly related to SWC and soil nutrients. At this time, soil clay formed a protective barrier for the survival of the dominant classes Sordariomycetes and Dothideomycetes and increased their relative abundance. In summary, the fungal community responded significantly to SWC on the southern shore of the Hulun Lake ecosystem in Inner Mongolia, China, and the fungal community composition of the HW group was stable and easier to survive.