Pleurotus tuber-regium (Fries) Singer (PTR), both an edible and a medicinal mushroom also known as tiger milk mushroom, has experienced growing popularity and economic importance due to its flavor, nutritive value, and medicinal effects. In this study, the antioxidant and antiangiogenic activities of a 60% ethanol extract (EE) obtained from the sclerotium of PTR were investigated. Typical phenolic compounds including protocatechuic, chlorogenic, syringic, ferulic, and folic acid were identified and quantified in EE by the HPLC-UV-ESI/MS analyses. EE possessed strong antioxidant activity and could dose-dependently inhibit vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cells (HUVEC) migration and tube formation. qPCR results showed that VEGF-induced FGF, ANG-Tie, and MMP gene expression as well as VEGFR were down-regulated at the mRNA level after treated with EE, suggesting that multiple molecular targets related to angiogenesis was involved. Furthermore, EE also inhibited the formation of subintestinal vessel plexus (SIVs) in zebrafish embryos in vivo. All of these suggested that EE of PTR could be the source of potential inhibitors to target angiogenesis.