Biological efficiency (BE), the ratio of fresh mushrooms harvested per dry substrate weight, expressed as the percentage of Lentinula edodes, also known as shiitake, was determined using the ‘Sanjo 701’ strain stored in the Department of Mushroom at the Korea National College of Agriculture and Fisheries. The mycelia were grown in glass columns with varying levels of moisture content and varying mixing periods of 0.5, 1, 2, and 3 hours. The substrate was sterilized using a steam pressure autoclave sterilizer at normal and high pressure to avoid contamination. The results showed that mycelial growth (126 mm/15 days) was optimized at 55% moisture content. The best mycelial growth of 117 mm/15 days was obtained with 2 hours of mixing time. Normal pressure sterilization yielded better results with mycelial growth of 96 mm/15 days at 100°C compared to 88 mm /15 days with sterilization at 121°C. Mycelial density was higher, i.e. 3(+++), with normal pressure sterilization compared to 2(++) with high pressure sterilization. Furthermore, sawdust mixed with 5% woodchips increased the substrate porosity and yielded higher mycelial growth. Thus, we demonstrated that the optimum harvest or potential increased yield of shiitake can be obtained by modulating moisture content, mixing time, and substrate porosity.