Abstract:[Background] The grape production in eastern Helan Mountains has the problems of poor soil nutrients, neglect of organic fertilizer application, and environmental pollution caused by the burning of fruit plant branches. [Objective] In view of the problems caused by long-term application of chemical fertilizers to the soil, we carried out field experiments to study the effects of fertilizer application and fungicide spraying on the soil physicochemical properties and the fungal community composition and diversity, aiming to underpin the sustainable and healthy development of wine grapes. [Methods] The physicochemical properties of the rhizosphere soil of ‘Cabernet Sauvignon’ were examined. Illumina MiSeq high-throughput sequencing was performed to determine the fungal community composition and diversity in the rhizosphere soils in seven treatments: conventional fertilization (CK), earthworm manure+fermented branches+100×fungicide (T1), earthworm manure+fermented branches+ 200×fungicide (T2), earthworm manure+fermented branches+300×fungicide (T3), earthworm manure+unfermented branches+100×fungicide (A1), earthworm manure+unfermented branches+200×fungicide (A2), and earthworm manure+unfermented branches+300×fungicide (A3). [Results] Compared with CK, other treatments significantly changed the chemical properties of the rhizosphere soil. Specifically, the fertilization treatments increased the organic matter, did not alter the soil pH, improved the soil structure, and activated the available nutrients in soil. Compared with CK, The number of fungal operational taxonomic unit (OTU) decreased in all treatments, and A2 treatment increased the fungal richness and diversity in the rhizosphere soil. Ascomycota, Basidiomycota, Chytridiomycota, and Mortierellomycota were dominant in all the seven treatments at the phylum level, accounting for 74.40%–86.97% of the total relative abundance. T2 and A2 treatments increased the grape yield by 19.34% and 14.72%, respectively. The correlation analysis showed that total nitrogen was the main factor affecting the fungal community structure; microorganisms were not significantly related to yield; electric conductivity and total nitrogen were factors closely associated with yield. [Conclusion] T2 and A2 treatments improved the soil microbial community structure and soil nutrients, which promoted grape growth and improved yield and production efficiency, providing a theoretical basis for the selection of suitable fertilization schemes for grapes.