The improvement of multi-resistance properties of the bacterial pathogen has recently been discussed as an emerging issue. In this regard, iron oxide nanoparticles have attracted the researchers’,attention due to their wide application in the realm of medicine. Iron oxide nanoparticles have a high specific surface area that enables them to interact with the bacterial surface structure and has considerable antibacterial activity. The current study aimed to synthesize a novel antimicrobial agent from iron oxide nanoparticles and determine its Minimum inhibitory concentration (MIC) on different gram-positive and negative variant bacterial strains isolated and characterized from the infected urinary tract of Iraqi elderly patients. This study was conducted from September 2020 to December 2020 on 75 urine samples collected from the infected urinary tract of elderly patients in the ages range of 60-75 years admitted to Al-Yarmouk Medical Hospital, Baghdad, Iraq. Isolation of bacterial isolates was carried out using differential and selective media. Afterward, they were characterized and confirmed using different biochemical tests and VITEK 2 system, respectively. Magnetic nanoparticles were fabricated by co-precipitation of ferric ions (Fe3+) and ferrous ions (Fe2+) in presence of ammonium hydroxide solution (25%). The characterization of synthesized nanoparticles was performed subsequently using UV-VIS spectroscopy analysis, Scanning Electron Microscope (SEM), Fourier transform infrared spectroscopy analysis, X-ray Diffraction analysis (XRD), and Energy-dispersive X-ray spectrum (EDX). The MIC of synthesized sonicated Fe3O4NP against different bacterial strains was determined using the broth culture dilution method through making serial dilutions of 50, 100, 200, 400, 500, 600, 800, 900 μ, g/ml from a 5mg/ml nanoparticle stock solution. Afterward, the lowest concentration of nanoparticles required to arrest the growth of bacteria was determined through the colony-forming unit of each treated bacteria on brain heart infusion agar. In total, 17bacterial isolates were identified from the infected urinary tract, five bacterial isolates (E. coli, Pseudomanas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, and Micrococcus luteus). In addition, two Proteus mirabilis strains were identified separately and were tested against synthesized Fe3O4NPto determine the MIC. The novel synthesized antibacterial agent showed excellent bioactivity, compared with controls (consisting of bacterial suspension without ferrous oxide nanoparticles), and the synthesized antibacterial agent was considered significantly active against all the bacterial strains at a p-value less than 0. 05. The Fe3O4NP were active against gram-negative more than gram-positive bacteria. The MIC of synthesized and characterized Fe3O4NPwasapplied on seven gram-positive and negative bacterial isolates using bacteria-Fe3O4NP complex. Significant effects were observed on all strains, compared with controls, and this complex could significantly inhibit gram-negative more than gram-positive bacteria.