BACKGROUND: The development of functional foods with health-promoting properties is a priority in addressing chronic diseases. Studying the effect of these foods on gut microbiota provides critical insights into the interplay between microorganisms, health, and disease. OBJECTIVE: This study aimed to evaluate the effects of fiber-enriched kombucha tea on biochemical parameters and gut microbiota composition in a healthy population. METHODS: A randomized, double-blind, parallel nutritional intervention trial was conducted with 60 participants (58 completed: 42 women and 16 men; mean +/- SD age: 39.79 +/- 14.58 years). Participants were randomized into two groups consuming either 250 mL/day of a control beverage (unfermented tea) or the fiber-enriched kombucha tea for six weeks. Lifestyle and biochemical data were collected, and gut microbiota composition and diversity were assessed using 16S rDNA sequencing. RESULTS: Significant biochemical and microbiota-related improvements were observed in the study group compared to the control. The treatment significantly affected triglyceride levels (p-value = 0.031). In particular, the study group exhibited a reduction in triglyceride levels very close to significance (baseline mean +/- SEM: 69.59 +/- 6.98 mg/dL; post-intervention: 62.80 +/- 5.14 mg/dL; p = 0.053), while the control group did not experience a significant variation. Additionally, fiber-enriched kombucha consumption led to a notable increase in Bifidobacterium abundance, recognized for its intestinal health benefits and immunomodulatory effects. A reduction in Ruminococcus torques, linked to inflammatory bowel diseases, was also observed. CONCLUSIONS: The studied fiber-enriched kombucha drink demonstrated potential health benefits, including triglyceride modulation and positive alterations in gut microbiota composition. These findings suggest its promise as a functional beverage for improving metabolic and gut health in healthy individuals. However, further randomized controlled trials are warranted to confirm these benefits. This trial was registered at clinicaltrials.gov as NCT06626997.