Lemon Basil Straw (Ocimum citriodorum Vis., LBS) is an alternative substrate for mycelia production of gray oyster mushrooms (Pleurotus sajor-caju (Fr.) Sing.). The LBS extracts were obtained by submerging the LBS in DI water at room temperature, 121°C, and 4°C. Mycelial growth was conducted in solid media, sorghum grains, and liquid media. The experiments were designed within completely randomized design with triplications. The results showed that the LBS extracted at the room temperature displays a high significant growth rate in solid media (12.92 mm²/day) and sorghum grain (less than 15 days of fully covered on the grains). The LBS extracted in 4°C was suitable for submerged condition for mycelial cultivation because the extract increased the mycelial growth and revealed highest amount of exopolysaccharides. A scale-up for 1-L mycelial production comparing between LBS 4°C extract and potato dextrose revealed significant differences (p<.05) in terms of biomass and exopolysaccharides contents. The study suggested that a guideline for improvement cultivation of the mycelial mushroom in the future.