This study investigated the ecological succession of prokaryotic and eukaryotic communities colonizing pristine polystyrene panels, used as model substrate, deployed for 25 weeks in an anthropogenically impacted environment, such a harbor. Using the eDNA metabarcoding targeting the 16S and 18S rRNA genes, we tracked the temporal dynamics of communities, highlighting shifts in biodiversity and community structure on plastic surfaces. The microbial biofilm assemblages demonstrated relative temporal stability, with Rhodobacteraceae (16.97%) and Flavobacteriaceae (17.99%) consistently dominant, confirming their ecological roles as pioneer and persistent taxa in biofilm formation on plastics. Eukaryotic colonization patterns reflected more pronounced succession, shifting from Alveolata (63.39%) and Stramenopiles (23.53%) during early stages to communities enriched in Chlorophyta (20.14%) and Opisthokonta (94.32%) over time. Alpha diversity, based on ASV richness, ranged from 1,875 to 2,481 for eukaryotes and 159 to 405 for prokaryotes, indicating dynamic succession of communities as part of natural processes. Notably, putative plastic-degrading prokaryotes were detected suggesting microbial adaptation and potential functional roles in polymer degradation. Finally, trophic profiling of the eukaryotic assemblages revealed a heterotroph-dominated system. The observed temporal changes reflect ecological succession modulated by local environmental stressors. Our findings underline the role of plastic debris as a different ecological habitat that harbor diverse microbial and eukaryotic assemblages including invasive or potentially harmful species, which could influence local biodiversity patterns and alter trophic interactions in marine ecosystems. These dynamics pose ecological risks and highlight the urgent need for improved plastic waste management strategies, particularly in semi-enclosed coastal systems, such as harbors, where limited water circulation promotes plastic accumulation with ecological implications.