Exudates (DE) secreted from two shiitake mushroom mycelia (strains 1358 and L5458) were evaluated for their antioxidative properties and phenolic content. 1358DE and L5458DE showed distinct antioxidant activity in different in vitro assays, including scavenging activity on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, hydroxyl radical, superoxide anions and hydrogen peroxide; the ability to chelate ferrous ions; reducing power; hemolysis inhibition activity in rat erythrocyte; and lipid peroxidation inhibition (IC50 values of 1358DE and L5458DE were 3.3 and 132.6; 44.5 and > 1000; 26.9 and 53.7; 153.6 and > 175.0; 176.0 and 521.0; 26.7 and 746.4; 47.8 and 736.9; and 3.1 and > 1000 microg/mL, respectively). Their total phenolic content was 237.33 and 24.08 mg gallic acid equivalent (GAE)/g of dry DE, respectively. Overall, these results show that 1358DE generally possesses better antioxidant properties than L5458DE, possibly due to its larger total phenolic content. Shiitake mushroom mycelial exudates, particularly of 1358DE, could be a good source of natural antioxidants.