“Plastisphere”, microbial communities colonizing plastic debris, has sparked global concern for marine ecosystems. Microbiome inhabiting this novel human-made niche has been increasingly characterized; however, whether the plastisphere holds crucial roles in biogeochemical cycling remains largely unknown. Here we evaluate the potential of plastisphere in biotic and abiotic denitrification and nitrous oxide (N2O) production in estuaries. Biofilm formation provides anoxic conditions favoring denitrifiers. Comparing with surrounding bulk water, plastisphere exhibits a higher denitrifying activity and N2O production, suggesting an overlooked N2O source. Regardless of plastisphere and bulk water, bacterial and fungal denitrifications are the main regulators for N2O production instead of chemodenitrification. However, the contributions of bacteria and fungi in the plastisphere are different from those in bulk water, indicating a distinct N2O production pattern in the plastisphere. These findings pinpoint plastisphere as a N2O source, and provide insights into roles of the new biotope in biogeochemical cycling in the Anthropocene