The bark of Polyalthia longifolia is traditionally reputed to lower blood pressure, stimulate respiration and help in fever and skin diseases, diabetes, hypertension and vitiated conditions of vata and pitta. Preliminary phytochemical investigation of various extracts of the bark of Polyalthia longifolia showed the presence of flavonoids, alkaloids, steroids and carbohydrates. An attempt has been made to isolate flavonoids and perform antioxidant and antimicrobial activity of the same. We carried out DPPH radical scavenging assay, nitric oxide scavenging assay, metal chelating activity and reducing power activity in antioxidant activity. In antimicrobial activity, we used six microorganisms, which included two Gram positive, two Gram negative bacteria and two fungi. Both the isolated flavonoids exhibited a concentration-dependant free radical scavenging capacity. Isolated compounds showed promising results against various micro-organisms in comparison with standard drugs (Penicillin, Gentamicin and Ketoconazol).