This study focused on the effects of vinasse (V), a by-product of the sugar-ethanol industry, combined with mineral nitrogen fertilizer (N) and straw retention on the fungal community diversity, composition, and structure in a sugarcane-cultivated soil. The experiment consisted of a combination of V, mineral N and sugarcane-straw blanket. Soil samples were collected at 7, 157, and 217 days after planting, corresponding to maximum carbon dioxide emissions from soil after three repeated applications of fertilizers into the soil. Across 57 soil metagenomics datasets, it was revealed that the application the V in combination with N fertilizer and straw retention decreased the diversity, evenness and richness of fungi at the community level in soil. Analysis of the soil fungal community composition based on the 20 genera most abundant revealed decrease for Blastomyces, Melampsora, and Penicillium after the third application of V in combination with N fertilizer and straw blanket. An opposite response was revealed for Amauroascus, Cantharellus, Chrysosporium, Clavaria, Morchella, Puccinia, and Tuber in soil under this treatment. Shifts in fungal community composition were followed by increases in mycorrhizal and decomposers soil-borne fungi and decrease in potentially pathogenic fungi, but not by changes in community structure. Based on these results, it is possible to attest that repeated applications of V in combination with N fertilizer and sugarcane-straw blankets affect ecological aspects of the soil fungal community composition and potential functions played by fungi in sugarcane soil, which are essentials to ecosystem function and sustainable management of agricultural ecosystems.