Metabolic alterations of Cordyceps bassiana mycelium were investigated under the following culture medium and light conditions: dextrose agar supplemented with 0.5% yeast extract (SDAY) medium with light (SL), SDAY medium without light (SD), nut medium without light (ND), and iron-supplemented SDAY medium without light (FD). The levels of asparagine, aspartic acid, glutamic acid, glutamine, histidine, lysine, ornithine, and proline were significantly higher under SD and SL conditions. The levels of most of the alcohols, saturated fatty acids, unsaturated fatty acids, fatty acid esters, sterols, and terpenes were higher under the ND condition than in the other conditions, but beauvericin was not detectable under the ND condition. The FD condition was favorable for the enhanced production of aminomalonic acid, malic acid, mannonic acid, and erythritol. Thus, the metabolic characteristics of C. bassiana can be manipulated by varying the cultivation conditions, rendering this fungus potentially favorable as a nutraceutical and medicinal resource.