| Summary: | The depletion of fossil fuels and the emission of greenhouse gases have increased the demand for new and sustainable energy sources, leading to growing interest in using fast-growing filamentous fungi as a source of bioenergy. This study aimed to optimize the production of exopolysaccharides (EPS) and mycelial biomass (MB) from the native medicinal mushroom, <i>Lignosus rhinocerus,</i> through submerged liquid fermentation. Using response surface methodology (RSM), it was found that the glucose concentration and speed of agitation significantly influenced the production of MB and EPS (<i>p</i> < 0.05), while the initial pH medium had an insignificant effect. The validated optimized parameters of 50.0 g/L glucose, initial pH 4.0, and 128 rpm for speed of agitation were tested in 500 mL shake flasks, 5 L, and 13 L stirred-tank (STR) bioreactors. The production of MB and EPS increased significantly by ~1.2-fold in the 5 L STR and further increased to ~1.7-fold (MB) and ~2.4-fold (EPS) in the 13 L STR bioreactor compared to the shake flask. The lipid content of MB was also determined, with a result of 2.07% <i>w</i>/<i>w</i> using the Soxhlet extraction method. To conclude, this study emphasizes the ability of <i>L. rhinocerus</i> as a new source of bioenergy through large-scale production, with optimized parameters serving as a reference for future research and practical applications.
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