<p>This study investigates the biodegradation of High-Density Polyethylene (HDPE) using a mixed microbial culture in a single-stage semi-anaerobic reactor, aiming to mitigate the environmental impact of plastic waste. Unlike previous research that primarily employed pure culture studies, our approach utilizes a mixed culture system, which can offer a broader range of enzymatic activities and potentially more efficient degradation. Additionally, the use of a semi-anaerobic reactor provides a more realistic simulation of environmental conditions compared to fully aerobic systems. In this study, petroleum oil is introduced as a supplementary carbon source to enhance microbial activity and facilitate the breakdown of HDPE. Over a 159-day period, it is observed that 13.6% reduction in HDPE mass, indicating partial polymer degradation. Scanning Electron Microscopy analysis revealed substantial surface alterations, including increased roughness and the formation of cracks, suggesting active microbial colonization and breakdown of the polymer. Carbon mass balance assessments demonstrated the efficient conversion of the polymer into microbial biomass. Furthermore, these assessments tracked a carbon loss of 11.8% from the HDPE sample, further supporting the efficacy of the degradation process.</p> Graphical Abstract <p></p>