The study aims to investigate the phytochemical composition, antioxidant potential, and anti-cancer activities of Ginkgo biloba L. leaf methanol extract. In this study, the phytochemical compounds, total phenolic and flavonoid contents of G. biloba leaf methanol extract were investigated. Additionally, antioxidant and anti-cancer activities [against HT-29 (human colon cancer line), HeLa (human cervical cancer line), and HEK-293 (human embryonic kidney cell line)] were assayed. The main phytochemical compounds were identified as gallic (0.94±0.01 mg/g), p-hydroxy benzoic (0.71±0.01 mg/g), and protocatechuic (0.60±0.02 mg/g) acids in G. biloba leaf methanol extract by HPLC. The total phenolic and total flavonoid contents were measured as 71.20±0.42 µg GAE/mg extract and 13.24±0.35 µg QE/mg extract, respectively. The high antioxidant activity was found in ABTS•+ assay (89.04±0.71%) while moderate antioxidant activity was observed in DPPH• (43.31±0.75%), metal chelating (49.04±0.49%), CUPRAC (absorbance: 0.85±0.01), and phosphomolybdenum (absorbance: 1.16±0.02) assays at 400 µg/mL. The IC50 values of G. biloba leaf methanol extract on HT-29, HeLa, and HEK-293 cell lines were recorded as 406.70±1.55, 84.86±0.98, and >800 µg/mL, respectively. The present study features a new addition to the antioxidant and anti-cancer properties of the therapeutically valuable G. biloba with its phytochemical content.