This study aims to clarify the role of variegatic acid (VA) secreted from Serpula lacyrmans in a chelator-mediated Fenton (CMF) system, including FeIII reduction and the generation of reactive oxygen species (ROS) in the presence of H2O2 and oxalate. As the principle component of the fungal extracellular matrix (ECM), β-glucan isolated from Basidiomycota species was also assessed in scavenging ROS with regard to its potential as a protective barrier for fungal hyphae. Our results demonstrate that VA was effective in reducing FeIII and promoting ROS generation. It is known that oxalate is necessary for solubilization of iron, but both iron reduction and ROS generation were impeded in the presence of oxalate. However, we observed that a higher pH (4.4) favored FeIII transfer from oxalate to VA to drive Fenton-based ROS generation as opposed to a lower pH (2.2), which would be found within the ECM. We propose that a pH-dependent FeIII transfer to VA is employed by S. lacyrmans to permit ROS generation within the higher pH wood cell wall, while limiting ROS production near the fungal hyphae. Further, β-glucan was found to scavenge ROS in acid environments and we postulate that this allows ROS scavenging within the ECM to further limit damage to the fungal hyphae when CMF reactions are active. Data support a role for the ECM in protecting fungal hyphae from ROS generated during decay processes and also support a potential role for a VA-mediated Fenton system in deconstruction of lignocellulose materials by S. lacyrmans.