The widespread contamination of the environment by microplastics (MPs), particularly polystyrene (PS) and polyethylene (PE), poses significant ecological and health concerns. While much attention has been given to the physical impact of MPs, their molecular interactions, particularly with genetic material, remain largely unexplored. This study examines how PS and PE MPs influence the structure and stability of human alternative i-motif (iM) DNA, which plays a key role in regulating gene expression and maintaining genomic integrity. Using biophysical techniques such as UV-Vis spectroscopy, circular dichroism (CD), FT-IR spectroscopy, and thermal melting analysis, our results have demonstrated the effects of PS-MPs and PE-MPs of different sizes on HRAS2 iM DNA. Our results reveal that PS-MPs, particularly those with a 100 nm diameter, strongly interact with HRAS2 iM DNA, causing hypochromism and shifts in the UV-Vis spectra, along with changes in secondary structure observed by CD. FT-IR spectroscopy confirmed alterations in DNA conformation, and thermal melting data showed increased stability of the HRAS2 iM DNA in the presence of PS-MPs. In contrast, PE-MPs exhibited weaker interactions, likely due to differences in size and surface properties. These findings suggest that PS-MPs, due to their small size, may disrupt the structural integrity of iM DNA, potentially affecting gene regulation. This work highlights the need to consider both the physical and biochemical properties of MPs in assessing their environmental and health risks.