Transition Metal Oxide (TMO) nanoparticles have emerged as promising materials for various applications including color imaging, magnetic recording media, soft magnetic materials, heterogeneous catalysis, and different field of biomedical science. Apart from the TMO, Fe₃O₄ nanoparticles hold great promise in a variety of biomedical uses such as drug delivery, cell separation, and MRI imaging. Magnetite (Fe₃0₄) nanoparticles exhibit their potential as antimicrobial agents due to their unique properties and interactions with microorganisms. This study focuses on the synthesis, characterization, and evaluation of the antimicrobial activity of magnetite (Fe₃O₄) nanoparticles prepared using the sol-gel method. The Fe₃O₄ nanoparticles were synthesized through a facile and cost-effective sol-gel route, involving the ferric nitrate and ethanol as precursors. Different characterization techniques, including Energy-Dispersive X-ray Spectroscopy (EDAX), X-ray diffraction (XRD), and UV-VIS NIR spectroscopy were employed to analyze the compositional analysis, crystalline structure, and optical properties of the nanoparticles. The EDAX and XRD analysis confirmed that the synthesized nanoparticles are near to stoichiometry and formation of single-phase magnetite nanoparticles. The obtained bandgap of synthesized nanoparticles is 5.03 eV. Furthermore, the synthesized Fe₃O₄ nanoparticles were evaluated for their antimicrobial efficacy against a panel of including both Gram-positive (e.g., Staphylococcus aureus) and Gram-negative (e.g., Enterobacter aerogenes) bacteria. Investigations into the nanoparticles biocompatibility and long-term effects would be crucial for their safe and effective utilization in real-world applications.