Trametes hirsuta is a medicinal mushroom that produces laccase. Its mycelium is a by-product when this species is used for laccase production. Aiming to develop its potential medicinal value, we investigated the chemical composition and immunomodulatory activity of T. hirsuta mycelia (THM). Dried THM contained 26.06% protein, 1.15% fat, 57.87% carbohydrates, and 5.47% ash. Sixteen free amino acids (2.63% total content) and 6 5'-nucleotides (adenosine 5'-monophosphate, cytidine 5'-monophosphate, guanosine 5'-monophosphate, uridine 5'-monophosphate, xanthosine 5'-monophosphate, and inosine 5'-monophosphate) constituting 0.275% were detected. Dominant sugars and polyols were fructose (2.47%), mannitol (2.03%), and glucose (1.8%); trehalose and arabinose contents were less than 0.10%. Evaluation of immunomodulatory activity in mice showed that THM could improve macrophage phagocytic function and serum hemolysin concentrations, but only the low-dose group significantly enhanced the natural killer cell activity and increased the spleen index, and only the middle-dose group remarkably increased the thymus index. Therefore, T. hirsuta mycelia could enhance immune function in mice and have immunomodulatory activity.