Centipeda minima is a Chinese herbal medicine used in the treatment of various diseases including cancer. An ethanol extract of the herb, its four fractions with different polarities, and two volatile oils prepared by steam distillation (SD) and supercritical fluid extraction (SFE) were investigated for their anti-angiogenic activity in a wild-type zebrafish model using a quantitative endogenous alkaline phosphatase (EAP) assay. The SFE oil displayed potent anti-angiogenic activity. Fifteen sesquiterpene lactones (SLs; compounds 1-15) isolated from the SFE oil were evaluated for their anti-angiogenic effect. Results revealed that pseudoguaianolide type SLs (1-8) inhibited vessel formation in the zebrafish embryos while guaianolide type SLs (9-15) showed little effect. Among the active ones, 6-O-angeloylenolin (1), a major component of SFE oil, possessed the strongest effect by reducing vessel formation in zebrafish embryos to 40% of the control value at 29.7 µM. Further study using the Tg (fli1a:EGFP) y1-type zebrafish model revealed that it blocked both intersegmental blood vessels (ISVs) and subintestinal vessels plexus (SIVs) formation in zebrafish embryos. Real-time polymerase chain reaction assay on the wild-type zebrafish embryos suggested that 6-O-angeloylenolin affected multiple molecular targets related to angiogenesis including VEGF receptor, angiopoietin, and its receptors. Taken together, our findings demonstrate that C. minima possesses anti-angiogenic activity, and 6-O-angeloylenolin is a promising candidate for the development of an anti-angiogenic agent.