The use of biogas to produce hydrogen is currently gaining more attention. One of the drawbacks for the valorization of biogas is the presence of H2S, a hazardous molecule that can cause damage in the metallic internal structures of industries. In this study, the H2S-removal performance of a fungi-based biofilter was investigated. First, an H2S-resistant fungal species was isolated from an industrial digestate and identified as Trichoderma harzianum. The capacity of this microorganism to metabolize H2S in a mineral medium was confirmed. Then, a bioreactor was constructed and put in place to monitor the elimination of gaseous H2S. A mix of cardboard, perlite, woodchips, and wood pellets was used as filling. Microbial development and the outlet gas composition were monitored during a 60-day experimental process during which H2S was completely removed. 97% of the introduced sulphur was detected in the used filling material (fungal species + packing material) by elemental analysis. 24% of the detected sulphur was identified by ion-exchange chromatography as SO42-. Elemental analysis, gas chromatography, and ion-exchange chromatography were used to determine the bioreactor sulphur balance. Metagenomic analysis underlined that H2S elimination was due to the presence of Trichoderma harzianum with a H2S-specific bacterial consortium.