Article InfoThe growing interest in mycelial biomass for its application in nurseries has led to the creation of inoculum production programs in New Zealand, Australia, the United States, and Chile, where the advantages of mycorrhizal inoculation have been demonstrated in plant morphological and physiological parameters. However, the mycelial biomass yields produced with these fungi through traditional submerged culture are generally low. Therefore, the aim of this study was to optimize the variables pH, temperature, and carbon source concentration required for maximum biomass production by the ectomycorrhizal fungi L. quieticolor and R. roseolus in liquid culture medium. The Box-Behnken (BB) design and response surface methodology (RSM) were used to establish the optimal conditions in the experimental area. Increases in biomass production of 1.2 g L -1 for 3.25 g L -1 for L. quieticolor (pH 5.5, 24°C and 20 g L -1 glucose) and 3.02 for 8.6 g L -1 for R. roseolus(pH 5.5, 28°C and 30 g L -1 mannitol) were found in comparison to the control culture (pH 5.5, 24°C and 10 g L -1 glucose) for both fungi. This optimization is the first step required to scale biomass production by L. quieticolor and R. roseolus in bioreactors.