Twelve new guaianolide-type sesquiterpenoids (1-12) and five known guaianolide derivatives (13-17) were isolated from an aqueous ethanol extract of the aerial parts of Chrysanthemum indicum. Their structures were determined through spectroscopic data analysis. The absolute configurations of the new compounds were assigned by X-ray crystallography and electronic circular dichroism. Compound 5 shows multiple cytotoxic activities against four human naso-pharyngeal carcinoma (NPC) cell lines (CNE1, CNE2, SUNE-1, and HONE-1) and one human intestinal epithelial cell line (HT-29) with IC50 values of 4.6, 6.0, 3.5, 4.3, and 9.6 μM, respectively. Compound 16 exhibits weak cytotoxicity against four NPC cell lines, CNE1 (IC50 = 7.3 μM), CNE2 (IC50 = 7.4 μM), HONE-1 (IC50 = 7.6 μM), and SUNE-1 (IC50 = 5.6 μM), but no cytotoxicity against HT-29 (IC50 > 10 μM).