Dioscorea glabra is a folk medicinal plant of India. The plant is used by the ethnic community of Tripura, India to treat different diseases. Present study was undertaken to determine the antispermogenic activity of Dioscorea glabra tuber and isolate few bioactive constituent. Dried tuber power was extracted using methanol and extract was fractionated to get pure compound. Antispermogenic activity of methanol extract and isolated fractions (1 mg/mL) were evaluated using in vitro assay method. Purification and characterization of fractions produced potent effect was carried out to identify the bioactive molecules. Extract and its two chromatographic fractions (T1 & T2) showed in vitro spermicidal activity as 30% and 33%, 37% sperm immobilization after 30 min respectively. Structural elucidation of these two fractions confirmed the constituents as triterpenoids. Present study proved that D. glabra tuber contains several spermicidal bioactive constituents. Future investigations could be useful to establish them as potent spermicidal molecule for human.