Porous activated carbons (AC-AN and AC-AO) for toluene adsorption were prepared starting from brewer’s grain biomass pretreated with microorganisms (Aspergillus niger van Tieghem for AC-AN and Aspergillus oryzae RIB40 for AC-AO). The structures and chemical properties of the three activated carbon materials (AC-AN, AC-AO, and AC that was not pretreated with microorganisms) were characterized by N₂ adsorption–desorption isotherms, scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), Raman spectroscopy, X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). The adsorption behavior of the three activated carbons for toluene was studied and correlated with the physical and chemical properties of these materials. The results suggested that the activated carbons prepared by microbial pretreatment had a rougher morphology, higher specific surface area, richer pore structure, fewer oxygen-containing functional groups on the surface, and better adsorption performance for toluene (increased by 31.5% and 18.3% with AC-AN and AC-AO, respectively) compared with the untreated activated carbon (AC). The Thomas model was used to fit the toluene adsorption data, indicating that the rich pore structure accelerated the kinetic process of toluene adsorption. Therefore, appropriate microbial pretreatment of the feedstock that is used to prepare activated carbon can effectively improve its adsorption capacity towards toluene.