Abstract: The work aimed to isolate the Pleurotus sp. from a bacteria contamination sawdust-based spawn. The number of bacteria was determined with the plate count assay. For the isolation of Pleurotus sp., the spawn was initially suspended in sterile water and then treated as follows: (1) vigorously mixed on a vortex mixer within five minutes; (2) kept continuously shaking at 180 r/min for 1-4 days; (3) kept to be static at 4℃ for 1-4 days. Next, the suspended spawn was collected by filtering using sterile gauze, and washed with sterile water. Finally, each slant was inoculated with a sawdust, allowing for growth at 25℃. As the control, the sawdust was directly inoculated into slants with one piece of sawdust per slant. The bacteria in the spawn determined were 15500 cfu/g. All the slants were found to be contaminated in the control. With the spawn vigorously mixed for 5 minutes before the isolation, no purified slants were observed. With shaking of 1, 2, 3 and 4 days, the success rate was 20%, 30%, 46.67%, and 63.3%, respectively. With kept to be static at 4℃ for 1, 2, 3 and 4 days, the success rate was 3.3%, 10%, 46.67%, and 60%, respectively. We conclude that mushroom strain can be effectively isolated and purified from bacteria contamination sawdust-based spawn using sawdust as inoculums by keeping spawn static or shaking in sterile water for 1-4 days before the isolation with the method of substrate mycelium isolation.