Kombucha is a prebiotic of non-dairy products. It is made from fermented plant-based infusion using using symbiotic culture of bacteria and yeast (SCOBY). During the fermentation process, yeast metabolize glucose in the substrat transforms into organic acid, vitamins, and microminerals. This transformation effect increases concentration and pharmacological activity. This research aimed to evaluate the antioxidant activity, vitamin concentration, and correlation between the kombucha process and increasing antioxidants of kombucha. The antioxidant activity was analyzed by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method and vitamin content was analyzed using spectroscopy UV Vis. Kombucha was prepared using five different substrates such as Etlingera elatior flowers, Senna alexandrina leaves, Syzygium malaccense pericarp, Sargassum crassifolium, and Kappaphycus alvarezii. The antioxidant activity, expressed as IC₅₀, was 27.930, 8.769, 25.132, 22.238, and 118.874 µg/mL, respectively, while the vitamin C contents were 7.97%, 6.92%, 1.568%, 1.51%, and 0.364%, respectively. Among the substrates evaluated, S. alexandrina kombucha exhibited the strongest antioxidant activity, as indicated by its lowest IC₅₀ value (8.769 µg/mL). Correlation analysis revealed a strong positive correlation between the antioxidant activity of the initial infusions and that of the resulting kombucha (r = 0.9656), indicating that substrates with higher initial antioxidant activity tended to produce kombucha with greater antioxidant activity. These findings demonstrate that substrate selection plays an important role in determining the antioxidant properties of kombucha and that S. alexandrina represents a promising substrate for producing kombucha with high antioxidant potential.