Cordyceps sinensis (C. sinensis), is a high altitude medicinal mushroom that is widely revered in traditional Oriental medicine for its energy enhancing and rejuvenating values. C. sinensis is known to possess several bioactive constituents like steroids, terpenoids, polyphenolics, glycosides, glycoproteins and peptides which impart therapeutic effects like antiproliferative, antihypercholesterolemic, immunomodulatory, prebiotic, etc. In the present paper, antioxidant fractions were prepared from aqueous extract ofIndian variety of C. sinensis by semi preparative high performance liquid chromatography (SP-HPLC). Fractionation was brought about on a Waters Sunfire® C18 column (10 mm x 250 mm), using 0.5% glacial acetic acid and acetonitrile (95:5, v/v) as the mobile phase, maintained at an isocratic flow rate of 5 mL/min. Eight fractions namely, F1 to F8 were collected in separate vials. Evaluation of total phenolic content (TPC) and total flavonoid content (TFC) confirmed significant antioxidant potential of these fractions. This observation was further substantiated by HPTLC fingerprinting that verified the presence of numerous antioxidant compounds in F1 to F8. Characterization by Fourier Transform Infrared (FTIR) spectroscopy, 1H and 13Cnuclear magnetic resonance (NMR) spectroscopy, and electrospray ionization mass spectroscopy (ESI-MS) manifested the presence of novel bioactive compounds in the fractions e.g.: n-heptanyl-beta-D-glucuronopyranosyl- (4'-1”)-beta-D-glucopyranosyl-(4”-1”') beta-D-xylopyronoside, 1-hexadecanyl glycerol, 1-pentadecanyl glycerol, myristinylxylosidetetradecanoylxyloside, noctadecanoic acid, arachidyl-beta-D-glucopyranosyl-(6'-1”)-beta-D-glucopyranosyl- (6”-1”')-beta-D-glucopyranoside, nonacosan-13-beta-ol and beta-D-glucopyranosyl- (6-1)-o-beta-D-glucopyranoside.