The Grape Valley of Turpan in Xinjiang is a renowned viticultural region located in an arid area of northwest China, characterized by a distinctive ecosystem shaped by high temperatures, drought, and saline-alkaline soils. Such unique environmental conditions are anticipated to harbor distinct microbial resources. However, the indigenous yeast communities and their functional potential in this region remain poorly explored. In this study, we integrated high-throughput sequencing of the ITS region from soil samples collected at eight representative sites with traditional culture-based methods to profile the fungal community and isolate cultivable yeasts for functional characterization. Sequencing results revealed that the soil fungal community was dominated by Ascomycota and Basidiomycota, with core genera including Pseudogymnoascus, Botryotrichum, Alternaria, and Aspergillus. The in situ yeast community encompassed at least 28 genera, with Naganishia as the most dominant. A total of 235 yeast strains representing 15 genera were successfully isolated. Functional screening indicated that a majority of the isolates produced industrially relevant enzymes such as beta-glucosidase, protease, and amylase. Several strains exhibited antagonistic activity against Gram-positive bacteria, and 54 strains showed resistance to anidulafungin. Following preliminary safety assessment, simulated fermentation assays using the selected non-Saccharomyces yeasts TW1-01 (Torulaspora delbrueckii) and TW2-03 (Pichia terricola) demonstrated their desirable oenological properties. Specifically, they contributed to enhanced glycerol production and increased color density in wine, while maintaining complete fermentation with alcohol content comparable to the control. These strains also demonstrated a robust capacity for autonomous malic acid degradation, efficiently converting it to lactic acid, and increasing the free radical scavenging rate by at least 20%. This study underscores the Grape Valley of Turpan as an important reservoir of fungal and yeast diversity, providing valuable microbial resources and a theoretical foundation for developing novel biocontrol agents or functional starters for the food industry.