<p>The present study investigates microbial hydrolysis as a viable approach for the biodegradation of Polyethylene terephthalate plastics to mitigate the environmental impact of its accumulation. It underscores the role of microbial degradation in reducing Polyethylene terephthalate persistence in ecosystems using a semi-anaerobic reactor. Petroleum oil serves as a supportive substrate, aiding in the adaptation of the hydrolysis system. The diversified microbial community in the anaerobic sludge was used for biodegradation, which could release the extracellular enzymes. Experimental findings reveal a considerable 11.5% degradation of Polyethylene terephthalate, confirming the efficacy of microbial hydrolysis in degrading this stubborn polymer. Additionally, Scanning Electron Microscopy analysis illustrates substantial morphological alterations on the Polyethylene terephthalate surface induced by hydrolysis reaction, providing important insights into structural modifications. The utilization of petroleum oil as a substrate introduces a novel dimension to enhance microbial enzymatic activity, further assessing the carbon utilization and conversion into suspended biomass by carbon mass balance analysis. Overall, this study elucidates the mechanisms involved in Polyethylene terephthalate biodegradation, laying a scientific groundwork for eco-friendly plastic waste management.</p>