The rapid emergence of multidrug-resistant bacteria has intensified the search for alternative antimicrobial materials capable of overcoming conventional antibiotic limitations. Among various inorganic nanomaterials, magnesium oxide (MgO) nanoparticles have attracted considerable attention owing to their chemical stability, biocompatibility, low toxicity, and remarkable antibacterial activity. In this study, Fe-doped magnesium oxide nanoparticles with the composition Mg₀.₉₄Fe₀.₀₆O were synthesized using a simple chemical preparation route followed by calcination at 550 °C. The influence of iron incorporation on the structural, optical, and antibacterial properties of MgO nanoparticles was systematically investigated. Fourier-transform infrared spectroscopy confirmed the formation of Mg–O bonds together with hydroxyl functional groups that facilitate surface interactions with bacterial membranes. UV–Visible spectroscopy revealed strong absorption in the ultraviolet region, while Tauc analysis estimated a direct optical band gap of approximately 3.96 eV, indicating the semiconductor nature of the prepared nanoparticles. Antibacterial activity was evaluated against the Gram-positive bacterium Staphylococcus aureus using the agar diffusion method. The synthesized nanoparticles exhibited a clear inhibition zone of approximately 15 mm, demonstrating effective antibacterial performance.