Hot-water extracted natural media were made from raw materials for mycelial culture of Pleurotus eryngii. Poplar sawdust, wheat bran and rice bran were used as substrates for hot water extraction. The mixed substrates of poplar sawdust, wheat bran, and rice bran with 50 : 20 : 30 (v/v/v, PWR523) and 50 : 30 : 20 (v/v/v, PWR532) were optimal for mycelial growth of P. eryngii, respectively. The hot-water extracted natural media from PWR523 and PWR532 showed a rapid mycelial growth and spawn running compared to PDA. There was no significant difference in mushroom yield when the mycelium grown on the hot-water extracted natural media was used as the inoculum source for producing fruit body.