Variation of metabolic profiles in Cordyceps pruinosa mycelia cultivated under various media and light conditions was investigated using 1H nuclear magnetic resonance (NMR) analysis and gas chromatography mass spectrometry (GC-MS) with multivariate statistical analysis. A total of 71 metabolites were identified (5 alcohols, 21 amino acids, 15 organic acids, 4 purines, 3 pyrimidines, 7 sugars, 11 fatty acids, and 5 other metabolites) by NMR and GC-MS analysis. The mycelia grown in nitrogen media and under dark conditions showed the lowest growth and ergosterol levels, essential to a functional fungal cell membrane; these mycelia, however, had the highest levels of putrescine, which is involved in abiotic stress tolerance. In contrast, mycelia cultivated in sabouraud dextrose agar with yeast extract (SDAY) media and under light conditions contained relatively higher levels of fatty acids, including valeric acid, stearic acid, lignoceric acid, myristic acid, oleic acid, palmitoleic acid, hepadecenoic acid, and linoleic acid. These mycelia also had the highest phenolic content and antioxidant activity, and did not exhibit growth retardation due to enhanced asexual development caused by higher levels of linoleic acid. Therefore, we suggested that a light-enriched environment with SDAY media was more optimal than dark condition for cultivation of C. pruinosa mycelia as biopharmaceutical or nutraceutical resources.