Kombucha is a potential probiotic tea-based drink with increasing worldwide consumption. Studies on this probiotic beverage are growing rapidly, especially about micronutrients and microbial population. As such, the present study performed the molecular identification of the microorganism and evaluated 5-methyltetrahydrofolate content by HPLC-DAD, phenolic compounds, flavonoids, carotenoids, antioxidant activity by spectrophotometric methods, and physicochemical composition of green tea kombucha on fermentation days 1, 3, 7, 14, and 21. DNA sequencing identified the Microbacterium genus as predominant. However, was unable to safely determine the species level because of the rRNA 16S gene sequence similarity between four species M. ureisolvens, M. yannicii, M. chocolatum e M. atlanticum. The concentration of 5-methyltetrahydrofolate found on the third day was 39.12 ± 1.32 μg/mL (liquid) and 45.78 ± 8.42 μg/mL (polymeric biofilm); On the twenty-first day it was 50.87 ± 3.56 μg/mL (liquid) and 54.88 ± 3.89 μg/mL (polymeric biofilm). Total phenolic compounds increased with fermentation; however, flavonoids and carotenoids were degraded by the process. The information on 5-methyltetrahydrofolate is unprecedented and highly relevant for food guidelines, since related deficiencies can lead to fetal malformation in the first three months of pregnancy. Lastly, the best fermentation time to obtain 5-methyltetrahydrofolate and others bioactive compounds is between days 7–14. Further analyses are also encouraged to understand the bioavailability of the vitamin.