Abstract: Different agricultural tillage practices create variable soil ecosystems that have different effects on soil microorganisms, especially those around the root system. Conservation tillage has numerous advantages and is used worldwide, but its sustainability has been challenged by the increase in soil pathogenic microorganisms caused by stubble mulching. To better understand the effects of long-term conservation tillage on the soil ecosystems in the dryland wheat regions of the Loess Plateau, this study used data from a 27-year conservation tillage experimental platform in Linfen, Shanxi Province, and high-throughput sequencing technology to investigate the response of the soil fungal community structure and diversity to different tillage treatments, including traditional tillage (TT1), no-tillage with stubble (NTS), and no-tillage with stubble after subsoiling (SNTS). The results showed that there were considerable differences in the composition and relative abundances of soil fungi among three treatments, and the NTS treatment included some unidentified or unknown fungi. Ascomycota, Basidiomycota, and Zygomycota were the dominant phyla of soil fungi in each treatment. Compared with TT1, the relative abundances of Ascomycota, Zygomycota, Chytridiomycota, and Glomeromycota increased under the two conservation tillage treatments, whereas the relative abundance of Basidiomycota significantly decreased. According to the heat map of species distribution at the genus level, the relative abundances of Gibberella and Fusarium increased compared to TT1 treatment, and Gibberella was the dominant genus under the two conservation tillage treatments. This indicated that long-term conservation tillage may increase the risk of wheat scab in this area. UPGMA (unweighted pair-group method with arithmetic means) showed that the soil fungal community composition was similar between the two conservation tillage treatments, which differed from the TT1 treatment. The key biomarker in the TT1 treatment belonged to Basidiomycota, whereas the key biomarkers belonged to Ascomycota for the NTS and SNTS treatments. The NTS treatment had the highest richness and PD_whole_tree of soil fungi, whereas the SNTS treatment had the highest diversity. However, there were no significant differences in the diversity and richness indices of soil fungi among three treatments. In conclusion, long-term conservation tillage significantly changed the structure and composition of soil fungal communities, and improved the richness and diversity of soil fungi with no significant differences among three treatments. Conservation tillage (for 27 years) may also increase the risk of wheat scab. Therefore, to minimize soil pathogens, appropriate stubble mulching frequency, no-tillage, conventional tillage or subsoiling rotation, and moderate shallow tillage should be examined in future soil management and conservation tillage studies. This study provides guidance for promoting conservation tillage and soil management in the dryland wheat regions of the Loess Plateau.