To minimize cultivation costs, prevent insect-pest infestation, and improve the production efficiency of thermophilic mushrooms, plant substrates obtainedfrom local areas in Cambodia were used for production of both spawn and mushrooms. In this experiment, different sawdusts different organic wastes and grain ingredients and analyzed for improvement of spawnproduction efficiency. Four thermophilic mushroom species, Pleurotus sajor-caju (oyster mushroom, Sambok), Ganoderma lucidum (deer horn shaped), Auricularia auricula (ear mushroom), and Lentinula edodes (shiitake), were used to identify efficient new substrates for spawn and mushroom production. Although the mycelia in the rubber tree sawdust medium showed a slightly slower growth rate (10.9 cm/15 days) than mycelia grown in grains (11.2 cm/15 days in rice seeds), rubber tree sawdust appeared to be an adequate replacement for grain spawn substrates. Th findings indicate that rubber tree sawdust, sugarcane bagasse, and acaciatree sawdust supplemented with rice bran and calcium carbonate could be new alternative the substrates for . Although sugarcane bagasse and rubber tree sawdust showed similarly high biological efficiencies (BE) of 60% and 60.8%, respectively, acacia tree sawdust exhibited relatively a low biological efficiency of 22.4%. However, it is expected that acacia sawdust has potential for the mushroom cultivation when supplemented with currently used sawdust substrates in Cambodia, because of its relatively low price. The price of the sawdust (20 kg sawdust= 6500 Riel or 1.6 USD) currently used was 6.5 times higher than the price of acacia sawdust (201000 Riel or 0.25 )USD). Therefore, utilization for acacia sawdust for mushroom cultivation could become feasible as it would reduce by producing costs of mushrooms in rural areas of Cambodia.