The alarming transmission rate of surgical site infections (SSI) in hospitals due to ineffective sterilization has encouraged researchers to search for a safe and easily available antibacterial agent. Common sterilization methods involving UV radiation and fumigants lead to hazardous effects on the environment and humans. These drawbacks have caused researchers to shift their attention towards visible light to activate certain materials to act as antibacterial agents. Thus, the present work reports optimization and antibacterial studies of sol-gel coupled ultrasound synthesis of photo-activated magnesium oxide (MgO) nanoparticles. The transmission electron microscope (TEM) and diffuse reflectance spectroscopy (DRS) analysis confirmed that smaller sized particles ranging from 13 nm-25 nm are formed with narrower bandgap of 2.54 eV (1 eV = 1.602 × 10 −19 J). The size reduction in the MgO nanoparticles narrowed their band gap, compared to previous results, which extends their absorptivity of light wavelength from UV (<400 nm) to the visible light region (400-550 nm). The disc diffusion antibacterial analysis optimized using response surface methodology (RSM) revealed that a 0.01 mol/L MgO nanoparticle concentration of 531 μL dosages exhibited a maximum zone of inhibition (ZoI) of 54.1 mm against E. coli, which was achieved with a visible light distance of 5.7 cm. Similarly, a maximum ZoI of 61.3 mm for S. aureus was obtained with a visible light distance of 5 cm and MgO concentration and dosage of 0.01 mol/L and 401 μL. This study confirms the ability of MgO nanoparticle as an alternate and better antibacterial agent via photoactivation for the first time. These photo-activated MgO nanoparticles will be beneficial in the possible inhibition of bacterial growth in surgical equipment, lab coats, or even as antibacterial paints in hospitals.