Now a days the best way to treat human diseases is by using bioactive compounds obtained from natural sources like plants, microbes etc. The present project was therefore planned for the isolation and purification of bioactive fractions from Cassia glauca Lam leaves. Crude methanol extract (Sample Name F14) was prepared by Soxhlet extractor which was further purified by column chromatography using silica gel 60 mesh (70-230) and fractions were eluted by using gradient method. Thirteen fractions (F1-F13) were collected using three different organic solvents (n-Hexane, ethyl acetate, methanol) mixed in different combinations. Crude extract and fractions obtained after purification were used to evaluate antibacterial, antifungal, antioxidant and cytotoxic activities. Antimicrobial activities against two Gram positive bacteria; S. aureus and B. subtilis, and two Gram negative bacteria; P. multocida and E. coli and four fungal strains; Aspergillusniger, Aspergillus ustilago, Fusarium solani and Alternaria alternata were performed by using well diffusion method. Antioxidant activity was performed by using DPPH free radical scavenging assay. The cytotoxicity was done by hemolytic assay. In our experiments the maximum zone of inhibition (25 mm) was shown by F7 (100 % Ethyl acetate) against E. coli and maximum zone of inhibition (19.7 mm) against A. niger was observed by F2 (n-Hexane and Ethyl acetate 10:1). Maximum antioxidant activity (57%) was shown by F14. In our studies F9 (Ethyl acetate: Methanol) showed maximum (64%) cytotoxic activity. Those fractions that showed all activities (antimicrobial, antioxidant and cytotoxic) were further analyzed by thin layer chromatography (TLC). Finally, statistical analysis was done. The results can further enhance the medicinal potential of Cassia glauca Lam leaves for the treatment of different diseases.