The increasing prevalence of microplastic pollution in aquatic environments has raised concerns about its impact on marine life. Among the different types of microplastics, polystyrene microplastics (PSMPs) are one of the most commonly detected in aquatic systems. Chaetoceros neogracile (diatom) is an essential part of the marine food web and plays a critical role in nutrient cycling. This study aimed to monitor the ecotoxicological impact of PSMPs on diatoms and observe enzymatic interactions through molecular docking simulations. Results showed that diatom growth decreased with increasing concentrations and exposure time to PSMPs, and the lowest photosynthetic efficiency (Fv/Fm) value was observed after 72 and 96 h of exposure to 200 mg L−1 of PSMPs. High concentrations of PSMPs led to a decrease in chlorophyll a content (up to 64.4%) and protein content (up to 35.5%). Molecular docking simulations revealed potential interactions between PSMPs and the extrinsic protein in photosystem II protein of diatoms, suggesting a strong affinity between the two. These findings indicate a detrimental effect of PSMPs on the growth and photosynthetic efficiency of diatoms and highlight the need for further research on the impact of microplastics on marine microbial processes.