Sclerotium rolfsii is a pathogenic fungus, infects about 500 plant species of about 100 families. Streptomyces species MTCC4 show remarkable activity against this fungus. The culture broth of Streptomyces species MTCC4 has inhibitory effect on the fungus in petriplate around the paper disc. Crude culture shows peaks at 2.8, 3.5, 3.8, 4.0, 5.4, 5.9and 8.43 min in HPLC analysis at the 220 nm wavelength at the flow rate of 0.5 ml/min, in C18 column. After extraction and purification of each fraction, the fraction with retention time 4.1 min at the Flow rate of 0.5ml/min and detection wavelength 310 nm, show maximum antifungal activity. 50% Methanol is used as mobile phase for HPLC analysis. Carbohydrate and protein test confirm the presence of carbohydrates and proteins. This compound is identified as a glycoprotein of 11.9 KD have antifungal activity against S. rolfsii.