Kombucha is a naturally fermented beverage with a rich history of development and popularity among consumers. The microbial ecology and complex microbial communities during the fermentation process are key factors that drive fermentation and shape the quality characteristics of kombucha. Kombucha features a symbiotic culture of bacteria and yeast that exists in two distinct phases within the drink: the liquid portion and the biofilm, which floats on the surface. Most investigations focused on describing the microbial community, biochemical properties, and association networks of the beverage independently. Recently, a mechanistic understanding of assembly of microbiomes in kombucha has emerged. This review aims to explore the evolutionary patterns of the complex microbiota in kombucha fermentation, the central metabolic pathways, the influence of environmental factors on community structure, and its regulation effects on the final flavor of kombucha. Furthermore, this review summarizes the impact of microbiota on the physicochemical properties (e.g., pH, ethanol, oxygen and sugar levels) and biochemical parameters (e.g., organic acids, polyphenolic compounds, and volatiles) of kombucha in a systematic manner. The potential health benefits and potential risks are evaluated associated with consuming kombucha or its bioactive compounds. On this basis, this review discussed the future development potential of kombucha and the limitations of related studies, aiming to provide valuable insights into the prospective scientific research and industrialization of kombucha.