This study investigates the synthesis and characterization of magnesium oxide (MgO) nano flakes (NFs) using pulsed laser ablation of magnesium ribbons. The resulting MgO NFs exhibit a flake-like structure with an average diameter of 100-400 nm and a wall thickness of 24 nm. Advanced analytical techniques, including X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), UV-visible spectroscopy, and energy-dispersive X-ray spectroscopy (EDX), confirmed the phase purity and functionality of the MgO NFs. The XRD analysis revealed a polycrystalline nature with dominant peaks at 2θ values of 38.86°, 59.46°, 62.83°, and 73.87°, corresponding to (111), (110), (220), and (311) diffractions, respectively. UV-visible spectroscopy showed a broad absorption peak, and Tau's formula yielded an energy band gap of 5.8 eV. FTIR spectroscopy detected Mg–O–Mg bending vibration, O–H stretching vibration, O=C=O stretching, and O–H bending vibration. The antibacterial activity of the optimized MgO NFs was evaluated against both gram-positive *Staphylococcus aureus* (S. aureus) and gram-negative *Escherichia coli* (E. coli) bacteria. The highest antibacterial activity was observed at a concentration of 200 μg/ml, resulting in inhibition zones of 15 mm ±0.5 mm for E. coli and 16 mm ±0.5 mm for S. aureus. The minimum inhibitory concentration (MIC) for both pathogens was determined to be 25 μg/ml, highlighting the promising antimicrobial potential of the MgO NFs. The study concludes that pulse laser ablation in liquid is an effective method for synthesizing MgO NFs with significant antibacterial properties, making them suitable for potential biomedical applications.This study investigates the synthesis and characterization of magnesium oxide (MgO) nano flakes (NFs) using pulsed laser ablation of magnesium ribbons. The resulting MgO NFs exhibit a flake-like structure with an average diameter of 100-400 nm and a wall thickness of 24 nm. Advanced analytical techniques, including X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), UV-visible spectroscopy, and energy-dispersive X-ray spectroscopy (EDX), confirmed the phase purity and functionality of the MgO NFs. The XRD analysis revealed a polycrystalline nature with dominant peaks at 2θ values of 38.86°, 59.46°, 62.83°, and 73.87°, corresponding to (111), (110), (220), and (311) diffractions, respectively. UV-visible spectroscopy showed a broad absorption peak, and Tau's formula yielded an energy band gap of 5.8 eV. FTIR spectroscopy detected Mg–O–Mg bending vibration, O–H stretching vibration, O=C=O stretching, and O–H bending vibration. The antibacterial activity of the optimized MgO NFs was evaluated against both gram-positive *Staphylococcus aureus* (S. aureus) and gram-negative *Escherichia coli* (E. coli) bacteria. The highest antibacterial activity was observed at a concentration of 200 μg/ml, resulting in inhibition zones of 15 mm ±0.5 mm for E. coli and 16 mm ±0.5 mm for S. aureus. The minimum inhibitory concentration (MIC) for both pathogens was determined to be 25 μg/ml, highlighting the promising antimicrobial potential of the MgO NFs. The study concludes that pulse laser ablation in liquid is an effective method for synthesizing MgO NFs with significant antibacterial properties, making them suitable for potential biomedical applications.