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Objective: Nanotechnology has been mentioned as a factor influencing science and industry. The use of nanotechnology and the production of nanoparticles have created new hope for solving human problems over the last few decades The purpose of this study was to biosynthesize MgONPs from leuconostic spp., evaluation MgO nanoparticles for antibacterial effectiveness against isolated Gram-negative bacteria that are resistant to several antibiotics. Methodology: Leuconostoc spp. has been used to synthesize magnesium oxide nanoparticles, we detected to MgONPs antibacterial activity, MIC, MBC, antibiofilm activity and in vivo cytotoxicity of MgONPs when injected in the rabbit at a concentration of 4 mg/ml and a volume of 1 ml per Kg of rabbit weight. Results: In the present study conclude that the spherical MgONPs synthesized by Leuconostoc spp., with an average diameter of 43.85 nm, showed antibacterial effects by using agar well diffusion as a test to determine the antibacterial activities of synthesized MgONPs against the Salmonella typhi MDR that are resistant to several antibiotics. The value of MICs for MgONPs (125 mg/ml) and MBC (500 mg/ml). MgONPs have antibiofilm activity against Salmonella typhi as measured by absorbance at 630 nm. When MgONPs were injected into rabbits, it caused hepatocyte degeneration and nuclei pyknosis (necrosis), as well as a decrease in the number of kupffer cells in the sinusoids. The examination of kidney sections from the tested group revealed swollen renal tubular epithelial cells and glomerular atrophy, necrosis generally includes cells welling and nuclear pyknosis. Conclusion: The results showed that the MgONPs have a wide range of activity to inhibit MDR bacteria and their biofilm formation, for this, we determine its toxicity. The results refer that the MgONPs is highly toxic to tissues such as the liver and kidney.