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Cordycepin, a nucleoside derivative, is an active component of parasitic fungus, Cordyceps militaris which has been known for many pharmacological activities. The present study was carried out to develop an extraction strategy for cordycepin and to evaluate its antimicrobial potential. Solvent- solvent extraction method was used to extract cordycepin from liquid culture of C. militaris. Crude concentrated extract of fermented broth was sequentially partitioned with hexane, chloroform and n-butanol. Further we assessed the effect of solvent-solvent ratio, extraction time and temperature on cordycepin extraction Antimicrobial properties of various Cordyceps militris derived fractions such as hexane; chloroform and n-butanol were investigated against human pathogenic microorganisms using the disc diffusion. Maximum yield of cordycepin was achieved at a solvent ratio of 1:2 (v/v) with extraction time of 90 minutes at 40°C. Final purity of the cordycepin was recorded up to 95% based on high performance liquid chromatography. Butanolic fractions, demonstrated the highest antimicrobial activity, due to the presence of cordycepin as an active component. The minimum inhibition concentration (MIC) of cordycepin was found to be 25-40 µg/disc for all the microorganisms tested. Hence Cordyceps militaris derived cordycepin could potentially be used in the treatment of infectious diseases caused by pathogenic microorganisms.