Bio-based materials are a relevant alternative to traditional building materials particularly due to their carbon sequestration and storage properties. However, their high flammability limits their use and the application of a fireproof treatment is needed. Since fireproof treatments may alter the fibers, and the acoustic properties depend on the material’s microstructure, it is essential to evaluate the impact of such treatments. The goal of this paper is to evaluate the performance of hemp fibers, before and after fireproof treatment. Also, it aims to understand the impact that it can have on their microstructure using acoustic parameters. The sound absorption of four treated formulations and one untreated reference configuration was evaluated with an impedance tube using a three microphones characterization method. This method allows the indirect determination of the characteristic parameters of the pore network within the samples using equivalent fluid modeling. In addition, the use of homogenization models such as Tarnow’s enables the estimation of the effective radius of the fibers in the samples. Thus, an increase in fiber radius was observed after fireproof treatment, providing a possible explanation for the experimentally observed decrease in airflow resistivity and viscous dissipative effects, as well as the reduction in acoustic absorption in the treated hemp fiber stacks.