The present study focuses on the green synthesis of copper sulfate (CuSO₄) nanoparticles using cassava (Manihot esculenta) starch as a natural stabilizing and reducing agent. The aim was to develop an eco-friendly synthesis route, identify the composition of the synthesized nanoparticles, and study their antimicrobial activity. Cassava starch offers a sustainable biopolymer matrix for nanoparticle formation due to its hydroxyl-rich polysaccharide structure. The synthesized nanoparticles were characterized using UV–Visible spectroscopy, Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and X-ray diffraction (XRD). The antimicrobial activity of the CuSO₄ nanoparticles was tested against Escherichia coli and Staphylococcus aureus using the agar well diffusion method. Results confirmed the formation of uniform, spherical nanoparticles with an average size range of 40–60 nm. The CuSO₄ nanoparticles demonstrated significant antimicrobial properties, suggesting potential applications in biomedical and agricultural fields.