This study reports the synthesis of magnesium oxide (MgO) nano flakes (NFs) via pulsed laser ablation of magnesium ribbons, investigating their potent antibacterial properties for biomedical applications. MgO NFs were characterized using advanced techniques, revealing a flake-like structure with an average diameter of 100-400 nm and a wall thickness of 24 nm. The laser ablation method produced high-purity MgO, confirmed by EDX and XRD analysis, which showed a polycrystalline structure with dominant peaks at 38.86°, 59.46°, 62.83°, and 73.87° corresponding to (111), (110), (220), and (311) diffractions. UV-visible spectroscopy indicated a broad absorption peak, and Tauc's formula yielded a band gap of 5.8 eV. FTIR analysis detected Mg–O–Mg bending vibration, O–H stretching vibration, O=C=O stretching, and O–H bending vibration. The MgO NFs demonstrated remarkable antibacterial efficacy against both gram-positive Staphylococcus aureus (S. aureus) and gram-negative Escherichia coli (E. coli) bacteria. Maximum 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 MgO NFs. The study also explored the antibacterial mechanisms of MgO NFs, including the production of reactive oxygen species (ROS) and the release of harmful ions, which disrupt bacterial membranes and inhibit microbial growth. The results indicate that MgO NFs are a promising alternative to antibiotics for antimicrobial applications.This study reports the synthesis of magnesium oxide (MgO) nano flakes (NFs) via pulsed laser ablation of magnesium ribbons, investigating their potent antibacterial properties for biomedical applications. MgO NFs were characterized using advanced techniques, revealing a flake-like structure with an average diameter of 100-400 nm and a wall thickness of 24 nm. The laser ablation method produced high-purity MgO, confirmed by EDX and XRD analysis, which showed a polycrystalline structure with dominant peaks at 38.86°, 59.46°, 62.83°, and 73.87° corresponding to (111), (110), (220), and (311) diffractions. UV-visible spectroscopy indicated a broad absorption peak, and Tauc's formula yielded a band gap of 5.8 eV. FTIR analysis detected Mg–O–Mg bending vibration, O–H stretching vibration, O=C=O stretching, and O–H bending vibration. The MgO NFs demonstrated remarkable antibacterial efficacy against both gram-positive Staphylococcus aureus (S. aureus) and gram-negative Escherichia coli (E. coli) bacteria. Maximum 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 MgO NFs. The study also explored the antibacterial mechanisms of MgO NFs, including the production of reactive oxygen species (ROS) and the release of harmful ions, which disrupt bacterial membranes and inhibit microbial growth. The results indicate that MgO NFs are a promising alternative to antibiotics for antimicrobial applications.