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<i>Cyperus rotundus</i> L. exhibits promising potential for the development of functional foods due to its documented pharmacological and biological activities. This study investigated the antioxidant and anti-diabetic properties of <i>C. rotundus</i> kombucha. The results demonstrated potent antioxidant activity with an IC₅₀ value of 76.7 ± 9.6 µL/mL for the DPPH assay and 314.2 ± 16.9 µL/mL for the ABTS assay. Additionally, the kombucha demonstrated alpha-glucosidase inhibitory with an IC₅₀ value of 142.7 ± 5.2 µL/mL. This in vitro antioxidant potential was further validated in vivo using <i>Drosophila</i>. <i>Drosophila</i> fed a high-sugar diet and supplemented with pure kombucha revealed significant increases in DPPH and ABTS free radical scavenging activity. <i>Drosophila</i> on a high-sugar diet supplemented with varying kombucha concentrations manifested enhanced resistance to oxidative stresses induced by H₂O₂ and paraquat. Concurrently, there was a notable decline in lipid peroxidation levels. Additionally, significant upregulations in CAT, SOD1, and SOD2 activities were observed when the high-sugar diet was supplemented with kombucha. Furthermore, in vivo assessments using <i>Drosophila</i> demonstrated significant reductions in alpha-glucosidase activity when fed with kombucha (reduced by 34.04%, 13.79%, and 11.60% when treated with 100%, 40%, and 10% kombucha, respectively). A comprehensive GC-MS and HPLC analysis of <i>C. rotundus</i> kombucha detected the presence of antioxidative and anti-glucosidase compounds. In conclusion, <i>C. rotundus</i> kombucha exhibits considerable antioxidant and anti-diabetic properties, demonstrating its potential as a beneficial beverage for health promotion.