The global rise in obesity is closely associated with the increasing prevalence of nonalcoholic fatty liver disease (NAFLD) and metabolic syndromes, posing significant health challenges. This study explored the ameliorative effects of Ophiocordyceps sinensis mycelia (TCM-NA01 formula: 1.4 mg adenosine and 55.2 mg polysaccharide/capsule) on hepatosteatosis development in a high-fat diet (HFD)-fed mice. TCM-NA01 supplementation significantly reduced (p <
0.05) body weight, adipose tissue, serum triglyceride (TG)/cholesterol (TC), aspartate aminotransferase (AST), and alanine aminotransferase (ALT), liver TG/TC levels in HFD-fed mice. Increased (p <
0.05) fecal-lipid and bile-acid outputs were observed. Apparent reductions in lipid-droplet and steatosis scores (p <
0.05) in the HFD-fed mice supplemented with TCM-NA01. Furthermore, TCM-NA01 modulated lipid metabolism by decreasing fatty acid synthesis and promoting fatty acid β-oxidation. TCM-NA01 also enhanced liver antioxidant capacity and decreased proinflammatory cytokines (p <
0.05). These findings underscore the potential of O. sinensis mycelia as a nutraceutical agent for alleviating hepatosteatosis, liver oxidative stress, and chronic inflammation, offering a promising strategy for the management of obesity and NAFLD.