Abstract: Environmental pollution has posed serious harm to human health. The use of adhesives has also caused in the traditional paperboard. It can be expected to develop the environmentally friendly mycelium paperboard. Among them, the hyphae can form clamp-shape connection structures for the nutrient reproduction and transfer. A natural biocomposite material can grow on the natural agricultural waste, due to the high dipole moment and hydrogen bonding between macromolecules. In this study, the combination of Ganoderma weberianum/corn straw was determined to design the single-factor cultivation conditions, such as the cultivation time, straw particle size, and fructose addition. A systematic investigation was also implemented to explore the effects on the mechanical properties, waterproof properties, and thermal decomposition performance of the hyphal paperboard formed by hot pressing. The optimal cultivation was obtained after orthogonal experiments. The experimental results indicate that the various factors were varied in the influence on the edgewise strength, flexural stiffness, and static bending strength and waterproofness of paperboard. Firstly, the flexural stiffness reached 16.63 mN/m, which was 21.2% higher than that of kraft study (13.10 mN/m), when the cultivation time was 30 days. Moreover, the water absorption (24 hours) also decreased from 3.92% to 1.27% with the increase of cultivation time. Secondly, previous studies showed that the addition of fructose, glucose, and sucrose increased the biomass growth rate of strains. The fructose was chosen as an exogenous nutrient. Once the mass fraction of 5% fructose was added, the mechanical properties were the best with a flexural stiffness of 18.87 mN/m, which was 30.5% higher than that of kraft study (13.10 mN/m). Thirdly, the particle size of substrate shared the significant impact on the appearance and properties of the material. Furthermore, the mycelium paperboard made of 0.165-0.250 mm particle size was the closest appearance and feel to kraft paper. At the same time, the mycelium paperboard made of 0.165-0.250 mm straw was the least prone to deformation and loosening during testing. The molding was significantly improved, compared with the rest components. In terms of mechanical properties, the edgewise strength, the static bending strength, and the flexural stiffness increased by 17.4%, 14.9%, and 57.3%, respectively. The influence level of various factors was also ranked in the descending order of the particle size, cultivation time, fructose addition amount. Once the particle size was 0.165-0.250 mm, the cultivation time was 30 days, and the fructose addition was 5.0%. The edgewise strength, static bending strength and flexural stiffness of prepared paperboard were 14.36 N/mm, 10.89 MPa, and 33.09 mN/m, respectively. The water absorption rate and water thickness expansion rate of paperboard after 24 hours of water absorption decreased to 0.86 and 0.59%, respectively, as the particle size decreased. The mycelium paperboard prepared by different single factors shared the same thermal decomposition, starting at around 150 ℃ and reaching its the maximum at 350 ℃. This finding can provide a strong reference basis to develop and apply the mycelium composite materials in the future.