Kombucha is fermented and produced with a biofilm called a symbiotic culture of bacteria and yeast, which is drunk all over the world for its beneficial effects on human health and energy levels. The metagenomic study of kombucha frequently detected microorganisms in proteobacteria, firmicutes, and actinobacteria. And also, yeast and fungi are Ascomycota and Basidiomycota is present in green leaf and sugarcane juice fermented kombucha. The kombucha extracts’ biological activities were assessed using pH, total phenolic content, antioxidant, antibacterial, and anticancer activity. Fermentation may enhance biological activity and the generation of bioactive substances. These results showed the pH −3.1 ± 0.2 and TPC −0.721 μg/mL of gallic acid equivalent. The antioxidant radicals scavenging activity of kombucha was evaluated by DPPH, ABTS, H2O2 and TAC. The bioactive chemicals identified by FT-IR and HR-LC/MS analysis of Kombucha totaled 45 components. The identified compounds were further move on to perform molecular docking study against gastric cancer target proteins 4H9M, 2DQ7 and 1TVO are binding with Nequinate compounds showing best LibDock scores 105.12, 114.49, and 108.97. So, this study suggests that knowledge can potentially active bioactive compounds are present in kombucha and it’s stimulated the mechanism of gastrointestinal transit. Additionally, the metagenomic analysis gives strength to understand the bacterial and fungal distribution and its molecular mechanism from Kombucha.