Aims: Cordyceps militaris is a medicinal mushroom from Ascomycota. The aims of this study were to explore and identify the chemical compounds extracted in the non-polar fraction of the mushroom and to examine the biological potential of this extract. Methodology and results: The n-hexane extract metabolites were identified using gas chromatography-mass spectrometry (GC-MS) and results revealed the presence of 37 compounds delivered from different chemical classes and were mainly comprised of fatty acids and their esters (72%), carboxylic acids and their esters (10.39%), and a sulphur compound (7.1%). The n-hexane extract recorded a promising antioxidant effect (80.9±1.5%) at 80 mg/mL total extract; potent cholesterol reduction activity (100%) was obtained after 96 h incubation by the total metabolites (4%). The cytotoxicity of the compounds revealed 50% cytotoxicity concentration (CC50) ˃ 1 mg/mL and anti-rotavirus SA-11 effect where inhibition of virus attachment and penetration into infected cells was recorded at 50% effective concentration (IC50) of 300±0.2 µg/mL. Conclusion, significance and impact of study: This study confirmed the impact of the fatty acids produced by C. militaris as bioactive metabolites.