IntroductionOphiocordyceps gracilis is an entomopathogenic fungus and a precious traditional Chinese medicine with similar medicinal properties to Ophiocordyceps sinensis. However, information on the metabolite profiles of natural O. gracilis and its cultures is lacking, which limits their utilization.ObjectiveThe metabolic variations and antioxidant activities of O. gracilis cultures and natural O. gracilis were analyzed to evaluate the nutritional and medicinal value of O. gracilis and its cultures.MethodThe metabolite profiles of O. gracilis cultures (fruiting bodies and aerial mycelia), natural O. gracilis, and natural O. sinensis were compared by LC‐MS/MS coupled with multivariate data analysis. Furthermore, their antioxidant activities were evaluated based on their DPPH•, ABTS•+, and •OH scavenging abilities.ResultsA total of 612 metabolites were identified, and the metabolic compositions of the four Cordyceps samples were similar, with differences observed in the levels of some metabolites. There were 126 differential metabolites between natural O. gracilis and natural O. sinensis, among which fatty acids, carbohydrates, and secondary metabolites are predominant in natural O. gracilis. Furthermore, 116 differential metabolites between O. gracilis cultures and natural Cordyceps were identified, with generally higher levels in O. gracilis cultures than in natural Cordyceps. O. gracilis cultivated fruiting bodies exhibited the strongest antioxidant capacity among Cordyceps samples. Additionally, 46 primary and 24 secondary differential metabolites contribute to antioxidant activities.ConclusionThis study provides a reference for the application of natural O. gracilis and its cultures in functional food and medicine from the perspective of metabolites and antioxidant capacity.