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In this study, the sources, abundance, and ecological implications of microplastic (MP) pollution in Volturno, one of the main rivers in southern Italy, were explored by investigating the MP concentration levels in sediments collected along the watercourse. The samples were sieved through 5- and 2-mm sieves and treated with selective organic solvents. The polymer classes polystyrene (PS), polyethylene (PE), polypropylene (PP), polyethylene terephthalate (PET), polycarbonate (PC), nylon 6 (PA6), and nylon 6,6 (PA66) were quantified using pyrolysis–gas chromatography–mass spectrometry (Py-GC/MS) and high-performance liquid chromatography (HPLC). Furthermore, a 16S rRNA metagenomic analysis was performed using next-generation sequencing in Ion Torrent™ to explore the bacterial taxonomy and ecological dynamics of sediment samples. The MPs were detected in all samples collected from the study area. PP and PET were the most abundant and frequently detected polymer types in the analysed samples. The total MP concentration ranged from 1.05 to 14.55 ppm (parts per million), identifying two distinct data populations: high- and low-MP-contaminated sediments. According to the Polymer Hazard Index (PHI), MP pollution was categorised as hazard levels III and IV (corresponding to the danger category). Metagenomic data revealed that the presence of MPs significantly affected the abundance of bacterial taxa; Flavobacteraceae and Nocardiaceae , which are known to degrade polymeric substances, were present in high-MP-contaminated sediments. This study provides new insights into the ecological relevance of MP pollution and suggests that microorganisms may serve as biomarkers of MP pollution. [Display omitted] • Microplastic (MP) quantification in sediments was achieved using Py-GC/MS and HPLC. • MP pollution was highly variable along the river continuum. • Bacterial taxonomic abundance in sediment was altered by MP contamination. • Microbial biomarkers of river sediment MP pollution dynamics were identified. [ABSTRACT FROM AUTHOR]