Abstract: In order to explore the effects of grass cultivation on soil physicochemical properties and fungal communities in tobacco fields, Astragalus sinicus, Trifolium repens and Trifolium pratense were used as materials to determine the changes of soil physicochemical properties in tobacco fields after grass cultivation. High-throughput sequencing technology was used to detect the composition and diversity of soil fungal communities in tobacco fields, and the agronomic traits of tobacco were investigated at the budding stage of tobacco. The results showed that the main agronomic traits of tobacco treated with grass cultivation were better than those of natural grass cultivation. Compared with artificial weeding, grass cultivation had no significant effect on the growth of tobacco, while it increased soil pH, organic matter content in the tobacco field, and enhanced the supply of soil nitrogen, phosphorus and potassium. Ascomycota, Basidiomycota and Mortierellomycota were the main species of soil fungal community structure. The results of Alpha diversity analysis showed that grass species was an important factor affecting Alpha diversity. The main effect of grass density was not significant, but the interaction effect between grass species and density was significant. The Chao1 index, Shannon index and Simpson index of soil fungal community were significantly reduced by 112.5 and 75 kg/ha treatments compared with artificial weeding treatment. The results of LEfSe analysis and NMDS analysis showed that there were significant differences in fungal community composition between grass cultivation and artificial weeding. The grass cultivation treatment increased the relative abundance of Humicola and reduced the relative abundance of harmful fungi such as Fusarium and Aspergillus in the soil. Correlation analysis showed that the soil organic matter content was significantly negatively correlated with the relative abundance of Aspergillus, and the total potassium content was significantly negatively correlated with Fusarium. It can be seen that tobacco field grass cultivation can improve the soil physicochemical properties and reduce the relative abundance of some harmful fungi, which is of great significance to the health of tobacco planting soil.