High-density polyethylene (HDPE) is a widely used organic polymer and an emerging pollutant, because it is very stable and nonbiodegradable. Several fungal species that produce delignifying enzymes are known to be promising degraders of recalcitrant polymers, but research on the decomposition of plastics is scarce. In this study, white rot fungus, TBB-03, was isolated and characterized for its ability to degrade HDPE under lignocellulose substrate treatment. Ash () wood chips were found to stimulate laccase production (activity was > 210 U/L after 10 days of cultivation), and subsequently used for HDPE degradation assay. After 90 days, cracks formed on the surface of HDPE samples treated with TBB-03 and ash wood chips in both liquid and solid states. Raman analysis showed that the amorphous structure of HDPE was degraded by enzymes produced by TBB-03. Overall, TBB-03 is a promising resource for the biodegradation of HDPE, and this work sheds light on further applications for fungus-based plastic degradation systems.