Earlier the process of drug research was confined to the empirical testing of a large number of compounds for a specific activity. The bioactive phytochemical flavonoid has focused on the current scenario for various pharmacological activities. Flavonoids are chief active constituents have been used to management of various human diseases. Rutin (quercetin-3- rhamnosyl glucoside) as the flavonoids display antiviral, anticancer, anti-inflammatory, and heart disease protective activities. Rutin by acting as antioxidants exhibited several beneficial effects, such as anti-inflammatory, anti-allergic, antiviral as well as an anticancer activity. Dihydrofolate reductase (DHFR, EC 1.5.1.3) is one of the enzymes active in the folate cycle which plays a central role in DNA synthesis. DHFR Inhibition is a key element in the treatment of many diseases, including cancer and AIDS related infections. A investigate for new selective inhibitors is motivated by the resistance to common drugs observed in the course of treatment. In this paper an attempt has been made to find new DHF inhibitor by molecular docking. The rutin strictly follows Lippinski’s rule of five, thus having very good drug score as well as drug likeness score. The present study reveals that rutin has good binding affinity for DHFR and this can be use for the inhibition purpose and thus good antibacterial and anti cancer activity along with other important activities could be obtained.