Kombucha is one of the fastest-growing fermented beverages in popularity worldwide due to the health benefits its regular consumption may provide, such as reducing cholesterol and blood sugar levels and combating oxidative stress, among others. However, there are still few studies evaluating the in vitro cytotoxic potential of the beverage against cancer cell lines. Therefore, we produced kombucha using green tea cultivated in the city of Registro, Brazil, characterized its phenolic profile using UPLC-MS^E, and tested the cytotoxic potential of the beverage against lung adenocarcinoma epithelial cells (A549), human colon carcinoma cells (HCT8), human liver cancer cells (HepG2), human umbilical vein endothelial cells (HUVEC), and normal human lung fibroblast cell lines (IMR90). The green tea kombucha presented 92 phenolic compounds, most of which were classified as flavonoids. The kombucha demonstrated a cytotoxic profile in all tested cancer cell lines, evidenced by growth inhibition (GI50), decreased cell viability (IC50), and lethal concentration (LC50). In line with this, we observed that the extract presented a selective index (A549 = 2.84; HCT8 = 1.74; HepG2 = 1.4; and HUVEC = 1.23) in relation to the normal cell line, as IMR90 was not affected by the treatment (IC50, IG50, and LC50 > 500 µg GAE/mL). It is believed that the flavonoids, which are the most abundant in kombucha, are capable of acting on crucial signaling pathways for tumor development. Our findings indicate that green tea kombucha is effective in promoting cellular health in vitro, and complementary studies with animal and human models will be crucial to confirm these tested biological activities.