The farming areas in karst peak-cluster depressions, where residents live densely and utilize the land more intensively, pose a great risk for rocky desertification, and are usually seen as focal regions of ecological conservation. Appropriate tillage modes can set the foundation for sustainable land use and ecological security in the area. This study was conducted at the Guohua Karst Ecological Experimental Station in southwest China (a typical farming area in the peak cluster depression), to evaluate tillage effects on soil fungal community composition, fungal functions, and physicochemical properties. Major types of croplands, including pitaya field (representing conservation tillage, HF), maize field (representing plowing tillage, MF), sugarcane field (representing minimum tillage+straw return, SF), and paddy field (PF), were studied in contrast to forestland (artificial forest transformed from cropland, FL) and grassland (rehabilitated naturally from abandoned farmland, AF). The results showed that:(1) by comparison with the rehabilitated-land modes (FL, AF), the HF, MF and SF not only reduced the soil pH, soil organic matter contents (SOM), and total nitrogen (TN) but also altered the particle size distribution (clay, silt, sand). (2) The HF, MF, PF and FL increased the abundance-based coverage estimator (ACE) index and Shannon index of soil fungal communities, while SF and AF decreased the α-diversity index. The α-diversity index exhibited the indices declined as the soil depth increased. (3) In the study area, the dominant fungal groups were Ascomycota, Basidiomycota, and Mortierellomycota. Tillage did not change the species of dominant fungal strains, but affected their relative abundance. (4) The analysis of β-diversity showed that soil fungal community structure of each tillage mode was unique, however similarity existed among the HF and rehabilitated-land modes. Soil fungal community structures at 0-10 cm soil layers are similar to those at 10-20 cm, but they were largely different from those of the 20-40 cm depth. The FL, SF and HF had more distinctive species (biomarkers), followed by MF, PF, and the least was AF. (5) Soil pH, C/N, N/P were important factors affecting soil fungal community structures. (6) The effect of tillage modes on soil fungal function was obvious, pathotrophic fungi and saprotrophic fungi in HF, MF, SF, PF soil were significantly higher than those of FL and AF, moreover, the content of saprotrophic fungi in SF was the highest, HF had the highest symbiotrophic fungi. The characteristics of fungal community structures were similar among the HF, FL and AF, suggesting that no-tillage was conducive to maintaining the natural properties of soil fungal community structures, and shows a positive soil ecological effect.