Six flavonoids, namely luteolin (1), epicatechin (2), kaempferol 3-O-β-glucoside (3), kaempferol 3-O-α-rhamnoside (4), procyanidin A2 (5) and rutin (6) were purified from the EtOAc-soluble extract of litchi leaf by column chromatography. Their structures were elucidated by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) evidences. Luteolin and kaempferol 3-O-α-rhamnoside were found from this plant for the first time. Procyanidin A2 exhibited high anticancer activities against human hepatoma HepG2 and human cervical carcinoma Hela cells. However, it had poor anticancer activities against human lung cancer A549 and human breast cancer MCF-7 cells. Luteolin, epicatechin, procyanidin A2 and rutin showed good antioxidant activities than butylated hydroxytoluene (BHT). The antimicrobial activity assay indicated that luteolin possessed the strongest antimicrobial activity against Staphylococcus aureus, Escherichia coli, Shigella dysenteriae, Salmonella and Bacillus thuringiensis. Epicatechin, procyanidin A2 and rutin showed relatively weak antimicrobial activities.