Three enantiomeric sesquiterpene lactones were isolated under the bioguidance of the suppression of hepatitis B virus (HBV) surface antigen (HBsAg) from Senecio species, a widely distributed Chinese medicinal herb traditionally used for the treatment of hepatitis B, dermatosis and inflammation. The anti-HBV activity of the purified compounds was measured; all of them showed suppressive activity on the expression of HBsAg and HBV e antigen (HBeAg) in the HepG2.2.15 cell line. Real-time quantitative PCR analysis showed that the studied compounds decreased the number of infectious virions released, but did not inhibit the intracellular HBV DNA. The results suggest that enantiomeric sesquiterpene lactones may possess the potential to work synergistically with other antiviral compounds for the treatment of HBV infection.