Five previously unreported terpenoids, together with fifteen known analogs, were isolated from a methanol extract of the roots and rhizomes of Nardostachys jatamansi. Their structures, including absolute configurations, were elucidated by spectroscopic data and electronic circular dichroism (ECD) spectra analyses, as well as single-crystal X-ray diffraction for crystalline compounds. Structurally, (4R,5S,6S,7R)-1(10)-aristolane-8,9-diacid is a novel 8,9-dicarboxylic acid derivative of aristolane-type sesquiterpenoid. (4R,6S,7R,10S)-10-Hydroxyguaia-1(5)- 6,7-epoxy-2-one is an undescribed analogue of nardoguaianone K, with a rare 6,7-epoxide group. (4R,5R,6R,8R)- 1(10)-Isonardosinone-8-ol-9-one-7,11-lactone is an isonardosinane-type sesquiterpene bearing a γ-lactone ring. Dinardokanshone F is a rare example of a sesquiterpene dimer from N. jatamansi connected by an oxo bridge. The isolates were evaluated for their cytotoxic activity against four human pancreatic cancer cell lines (CFPAC-1, PANC-1, CAPAN-2 and SW1990). Compound epoxynardosinone exhibited significant cytotoxicity against CAPAN-2 cell lines with IC50 value of 2.60 ± 1.85 μM. 1-Hydroxylaristolone displayed comparable cytotoxicity on CFPAC-1 cell lines (IC50 1.12 ± 1.19 μM), compared to Taxol (IC50 0.32 ± 0.13 μM). 1-Hydroxylaristolone, 1 (10)-aristolane-9β-ol, 1(10)-aristolen-2-one, alpinenone, valtrate isovaleroyloxyhydrine and nardostachin displayed stronger cytotoxicity against PANC-1 cell lines with IC50 values ranging from 0.01 ± 0.01 to 6.50 ± 1.10 μM. 1(10)-Aristolane-9β-ol, 10-hydroxyguaia-1(5)-6,7-epoxy-2-one, nardoguaianone K, nardonoxide, epoxynardosinone, 1(10)-isonardosinone-8-ol-9-one-7,11-lactone, valtrate isovaleroyloxyhydrine and nardostachin showed remarkable cytotoxicity against SW1990 cell lines with IC50 values ranging from 0.07 ± 0.05 to 4.82 ± 6.96 μM. Furthermore, the primary mechanistic study of nardostachin demonstrated that it induced cell apoptosis via the mitochondria-dependent pathway, and induced SW1900 cell arrest at G2/M phase.