Kombucha has become a flavored tea drink fermented with a symbiotic community of fungi, yeast, and bacteria. Kombucha has been reported to show functional medicinal importance attributed to the presence of some critical metabolites and bioactive compounds. We developed kombucha on three different substrates like black tea, green tea, and Psidium guajava (guava) juice extract. Kombucha enriched with guava extract showed maximal growth in terms of weight (fresh and dry) and moisture percentage. Further, the total phenol, total flavonoid, and characterization of different phenolic compounds like gallic acid, catechin, guaiacol and coumaric acid with guava juice extract fermented kombucha exhibiting maximal production. Also, the high DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) radical scavenging activity over the control. The kombucha tea fermented on guava extract showed significant antimicrobial activity against eleven human pathogenic bacterial strains and four pathogenic fungi. From the current study, we conclude that the preparation of kombucha tea using guava juice extract might be used as a supplementary food product due to its high nutritional value.