The flavor development of koumiss is intricately linked to its starter culture. This research aimed to delve into the microbial diversity of mare milk and koumiss, isolate the dominant lactic acid bacteria (LAB) and yeast, and assess the effects of composite strains on the fermentation characteristics and flavor quality of koumiss. In mare milk, the dominant microbial genera were Enterobacter and Rhodotorula. As fermentation progressed, the microbial diversity of mare milk gradually declined, and Lactobacillus and Dekkera became the dominant bacterial genera. A total of 42 LAB types and 24 yeast types were isolated. A 1:1 combination of Lactobacillus helveticus 3-4 and Kazachstania unispora A-3 led to a high viable bacterial count and rapid acid production in koumiss. Twenty-one flavor substances were detected, endowing the koumiss with intense umami and sour profiles. This study offers a theoretical foundation for the development and industrial application of koumiss starters.