Bioassay-directed isolation and purification of the crude extract of Withania coagulans, using two assays for cancer chemopreventive mechanisms, led to the isolation of three new steroidal lactones, withacoagulin G (1), withacoagulin H (2), and withacoagulin I (3), along with six known derivatives (4-9). The structures and absolute stereochemistry of these compounds were determined on the basis of spectroscopic analyses, including 1D and 2D NMR, mass spectrometry, and CD analyses. The structure of 1 was confirmed using X-ray diffraction methods. Compounds 1-9 inhibited nitric oxide production in lipopolysaccharide-activated murine macrophage RAW 264.7 cells with IC(50) values in the range of 1.9-38.2 μM. Compounds 1 and 2 were the most active (IC(50) 3.1 and 1.9 μM, respectively). Withanolides 1-9 exhibited inhibition of tumor necrosis factor-α (TNF-α)-induced nuclear factor-kappa B (NF-κB) activation with IC(50) values in the range of 1.60-12.4 μM.