ABSTRACTCordyceps militaris, a medicinal fungus, has gained considerable attention owing to its potential health benefits, notably the production of bioactive compounds such as cordycepin. Cordycepin possesses significant antifungal, antibacterial, and antiviral properties. The present study focused on optimizing the fermentation conditions for C. militaris to boost the production of mycelia and cordycepin, alongside investigating its antifungal properties using in silico and in vitro approaches. The optimal conditions, yielding the highest cordycepin and mycelial biomass, were a temperature of 20°C and a pH range of 4–6, with glucose and sucrose as carbon sources and yeast extract and casein hydrolysate as nitrogen sources. Under these conditions, cordycepin production peaked at low pH (600–1000 mg/L) and with carbon and maltose (400–500 mg/L). The low temperature favored cordycepin production (400 mg/L), whereas casein hydrolysate as a nitrogen source boosted cordycepin yield (600 mg/L). The docking analysis indicated that cordycepin had the highest binding affinity for the tubulin beta chain 2 (−10.4 kcal/mol) compared to the fungicide tebuconazole (−7.9 kcal/mol for both targets). The in silico results were corroborated by in vitro studies, where the mycelial extract of C. militaris inhibited approximately 75% of fungal growth at a concentration of 6000 ppm. These findings suggest that optimizing fermentation conditions significantly enhances cordycepin production, and cordycepin shows antifungal solid activity, making it a promising agent for biocontrol in agriculture.