The aim of the present investigation deals with biological evaluation of Medicago lupulina leaves. For this purpose, different biological assays of methanolic extract (Crude) and its fractions that are chloroform fraction, n-hexane fraction, Ethyl acetate fraction, n-butanol fraction and aqueous fraction were carried out. The results from the agar diffusion method indicated that Crude showed maximum antibacterial activity against Staphylococcus aureus with the inhibition zone (29.02±0.18mm),

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... reas chloroform fraction also showed strong activity against Staphylococcus aureus (26.02±0.04mm). On the other hand, Crude showed maximum activity against Candida albicans and Candida glaberata with % inhibition of (66.02%±0.2) and (72.16%±0.09) respectively and Chloroform fraction showed good activity against Candida glaberata with % inhibition of (62.03%±0.09). Furthermore, Crude showed maximum insecticidal % mortality against Tribolium castaneum with (86%) mortality whereas (75%) mortality against Rhyzopertha dominica and Chloroform fraction showed maximum activity against Tribolium castaneum with (70%) mortality. Moreover, Crude showed tremendous antitumor activity with 89.40% inhibition and Chloroform fraction showed moderate level of tumor inhibition with 62.16% inhibition. Furthermore, the phytochemical estimation of Crude and its fractions showed the presence of Alkaloids, Flavonoids, Phenols, Tannins and Diterpenes.