The occurrence and development of tumors rely on the nutritional supply from blood vessels, which also serve as the main pathway for tumor metastasis. Inhibiting angiogenesis is one of the main strategies for cancer treatments. Chiral drugs, encouraged and inspired by chiral natural products, make up a major portion of marketed drugs. However, as an important source for synthetic chemistry and drug discovery, the counterpart of chiral natural products, the enantiomers, has received little attention. Here, we constructed a compound library containing 100 racemates (±)-1–100 and 4 pairs of enantiomers (33, 48, 59, 68) of 3,4-dioxygenated-4-aryl-quinolin-2(1H)-one alkaloids. Through extensive activity screening, we found that the compounds with 3R, 4R configuration, opposite to the natural products, exhibited potent angiogenesis inhibitory activity in zebrafish, while the 3S, 4S-configured natural derivatives have no effects. More importantly, compound (+)-48, named as (3R, 4R)-CHNQD-00728, significantly inhibited hepatic tumor growth in doxycin hydrochloride-induced liver-specific enlargement zebrafish. Examining the phenomenon and unlocking dark matter of enantiomeric natural products in innovative drugs discovery can provide a new perspective on organic synthesis and medicinal chemistry, thus enabling a broader exploration.