In order to explore the effects of applying distillers’ grain (DG) organic fertilizer on tobacco growth and soil microbial community, a field experiment with the application of DG organic fertilizer at rates of 0 and 1.5 t/hm² was conducted, and the effects of DG organic fertilizer on soil fungal diversity, fungal community composition and structure were investigated. The results showed that DG organic fertilizer increased the contents of soil organic matter and total carbon, and had no significant effects on the Chao1 and Shannon indices but altered the compositions of soil fungal communities at different tobacco growth periods (including rosette, vigorously growing and mature stages). Applying DG organic fertilizer increased the relative abundances of Mortierellomycota and Rozellomycota phyla at the resettling stage as well as the genus of Talaromyces and Rhodosporidiobolus. Soil available phosphorus was the dominant factor driving the changes in soil fungal community composition and structure.