The current study focused on the anticancer and antimicrobial potentials of the ethyl acetate extract of Pleiogynium timorense leaves and the characterization of its phytoconstituents. Ethyl acetate extract of pleiogynium timorense leaves showed a potent cytotoxic activity against ovarian cancer cell line (SKOV-3) with IC50 of (7.13 μg/mL) and a moderate activity against liver cancer cell line (HEPG-2) with IC50 =20.42 μg/mL , while it possessed a weak activity against Prostate cancer cell line (PC-3) (IC50 >100 μg/mL). In addition, the extract exhibited reasonable antibacterial activity against Staphylococcus aureus, Bacillus cereus, and Escherichia coli with MIC values of (200, 500 and 500 mg/ml), respectively without the detection of any activity against Salmonella typhimurium, Candida albicans and Aspergillus brasiliensis. The phytochemical study revealed the identification of eleven polyphenolic compounds representing 80.8 % of the total area; the major compounds were chlorogenic acid (40.8 %), gallic acid (11.1%), catechin (8.2%) and taxifolin (7.6 %). Moreover, gallic acid, kaempeferol and catechin were isolated and identified for the first time from the ethyl acetate extract. In addition, the plant extract is found to be rich in natural pigments (total carotenoids = 864.64 g/100g). In conclusion, the current study revealed that ethyl acetate extract of Pleiogynium timorense leaves is rich with polyphenolic compounds, which play a vital role in the plant bioactivities.