<p>The present work studied the role of the laser wavelength in the physical and biological properties of MgO nanoparticles synthesized with the Pulsed Laser Ablation in Liquid (PLAL) technique. X-ray diffraction indicates the crystal structure nature of MgO nanoparticles with an average crystallite size of 12 nm, synthesized with 532 nm. The Field Emission Scanning Electron Microscopy (FE-SEM) images show the semi-flake shape with average diameters of 31 nm for a laser wavelength of 1064 nm and 36 nm for a wavelength of 532 nm. Optical qualities were investigated using UV–Vis spectroscopy. This technique shows variations of the absorption edge and differences in the optical band gap, which could refer to the effect of the laser wavelength on particle size and shape. The energy band gaps were 3.5 eV and 3.8 eV for the samples synthesized with 1064 and 532 nm laser wavelengths, respectively. This work used Raman spectroscopy to study the vibrational modes, which showed characteristic peaks related to MgO NPs and bonds of magnesium oxide. Another important information obtained through the use of this technique concerns the influence that laser parameters have on the phonon dynamics of the nanoparticles. These optical and spectroscopic results indicated the modulation of properties in MgO NPs, which was achieved by changing the wavelength of the laser used. Biological tests revealed that MgO NPs can inhibit the growth of <i>S. aureus</i> and <i>E. coli</i>. These differences in biological performance were correlated with changes in structural properties. The effectiveness of the particles was much higher at the primary wavelength compared to the secondary wavelength, which made the particles even smaller. This work should aim to point out the importance of a laser wavelength as the basic parameter in the process by creating the multifunctional features of MgO NPs for advanced applications in materials science and biology.</p>