Microbial electrochemical technologies are promising for simultaneous energy recovery and wastewater treatment. Although the inhibitory effects of emerging pollutants, particularly micro/nanoplastics (MPs/NPs), on conventional wastewater systems have been extensively studied, the current understanding of their impact on microbial electrochemical systems is still quite limited. Microplastics are plastic particles ranging from 1 μm to 5 mm. However, nanoplastics are smaller plastic particles ranging from 1 to 100 nm. Due to their smaller size and greater surface area, they can penetrate deeper into biofilm structures and cell membranes, potentially disrupting their integrity and leading to changes in biofilm composition and function. This study first reports the impact of polystyrene nanoplastics (PsNPs) on syntrophic anode microbial communities in a microbial electrolysis cell. Low concentrations of PsNPs (50 and 250 μg/L) had a minimal impact on current density and hydrogen production. However, 500 μg/L of PsNPs decreased the maximum current density and specific hydrogen production rate by ∼43 % and ∼48 %, respectively. Exposure to PsNPs increased extracellular polymeric substance (EPS) levels, with a higher ratio of carbohydrates to proteins, suggesting a potential defense mechanism through EPS secretion. The downregulation of genes associated with extracellular electron transfer was observed at 500 μg/L of PsNPs. Furthermore, the detrimental impact of 500 μg/L PsNPs on the microbiome was evident from the decrease in 16S rRNA gene copies, microbial diversity, richness, and relative abundances of key electroactive and fermentative bacteria. For the first time, this study presents the inhibitory threshold of any NPs on syntrophic electroactive biofilms within a microbial electrochemical system.