Three previously unknown steroidal saponins named as govanoside C-E (1-3) along with four known compounds, govanoside B (4), protodioscin (5), 20β-hydroxyecdysone (6), and polypodine B (7) have been isolated from the rhizomes of Trillium govanianum Wall. ex D.Don. The structures of isolated compounds were elucidated by detailed analysis of 1D and 2D NMR, mass and IR spectroscopic data. Compounds 1 and 2 contained a rare sugar moiety i.e. 6-deoxy allose, while compound 3 has acetylated rhamnose moiety in its glycone part. Acid hydrolysis of new compounds followed by derivatization for GC analysis of glycone moieties was carried out for the confirmation of monomer saccharides present in each molecule. In addition, we have developed a protocol for the isolation of major steroidal saponin present in the rhizomes of Trillium govanianum i.e. borassoside E in gram scale. Parent extract, fractions and all pure molecules were screened to evaluate their antagonistic effects on acetylcholinesterase activity. Among extract and fractions, water fraction (IC50 value: 90.2 μg/ mL) was found most active whereas among pure molecules govanoside E (3) (IC50 value: 8.62 μM) was found most active against acetylcholinesterase. The molecular docking analysis was also carried out to further study the molecular interactions and binding free energy of the pure molecules with acetylcholinesterase.