Abstract: Aim of the study is to isolate, identify and evaluate the biologically active constituents present in the fruits of Durio zibenthinus Linn. The fresh fruit pulp of Durio zibenthinus Linn. were successively extracted by using various solvents like petroleum ether, chloroform, ethyl acetate and aqueous alcohol. The petroleum ether extract of the dried fruit pulp of the plant were subjected to isolation of phytoconsituents by counter current extraction and isolated compounds (β-Sitosterol and 3-β-hydroxy-21-Normethyl-19-vinylidenylursane) were partially characterized by using 1H NMR, 13C NMR and Mass spectroscopy. The petroleum ether extract and isolated compound (3-β-hydroxy-21-Normethyl-19-vinylidenylursane) were screened for aphrodisiac activity. The sexually active male mice were administered, the test or the solvent control doses, daily, orally, in their respective concentrations. The activities of male mice in each group were recorded individually for 60 min after 30 min of the drug administration on 1st, 7th and 14th day treatment including sperm count. In conclusion, out of the four extracts of the fruits of Durio zibenthinus Linn., Petroleum ether extract was subjected to isolation. From the above, two compounds were isolated. The 400 mg kg-1 b.wt. dose of petroleum ether showed significant aphrodisiac activity than all other treated dose of isolated compound (3-β-hydroxy-21-Normethyl-19-vinylidenylursane). Hence, this reveals that most herbs rely for their effects on variety of constituents. Further research is needed to identify biologically active constituents for fertility enhancing activity.