One of the most concerning issues related to global plastic production is the widespread presence of microplastics (MPs) in natural ecosystems. These recalcitrant micropollutants pose ecological and health risks. Biodegradation offers a promising approach to mitigating MP pollution. In aquatic environments, the most common microplastic type is polyethylene. This work investigates the ability of the bacterial strain Comamonas testosteroni CECT 326 to degrade high-density polyethylene (HDPE) MPs evaluating different process parameters. The results showed a reduction in polymer mass within 8 days, along with chemical and structural changes, indicating that the HDPE served as a carbon source for the bacteria. Temperature, MP size, and concentration influenced the degradation efficiency. ImageJ software analysis revealed a decrease in MP particle diameter and an increase in particle count, likely due to microbial activity fragmenting the MPs. High-Performance Liquid Chromatography-Mass Spectrometry (HPLC–MS) analysis of the culture medium before and after degradation identified the appearance of new compounds, some of which may be hazardous. This study confirms the potential of C. testosteroni to degrade HDPE MPs, contributing to mitigate the environmental impact of MP accumulation. However, it also underscores the need to address the release of toxic byproducts during biodegradation, which could lead to long-term adverse effects on ecosystems and human health.