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The <i>Paeonia emodi</i> (<i>P. emodi</i>)-mediated iron oxide nanoparticles (Fe₂O₃ NPs) were screened for in-vitro and in-vivo antibacterial activity against the <i>Staphylococcus aureus</i> (<i>S. aureus</i>) (ATCC #: 6538) and <i>Escherichia coli</i> (<i>E. coli</i>) (ATCC #:15224). The synthesized Fe₂O₃ NPs were characterized via nitrogen adsorption-desorption process, X-ray diffractometer (XRD), transmission and scanning electron microscopies (TEM and SEM), energy dispersive X-ray (EDX) and Fourier transform infrared (FTIR) spectroscopies. The S_BET was found to be 94.65 m²/g with pore size of 2.99 nm, whereas the average crystallite and particles size are 23 and 27.64 nm, respectively. The 4 μg/mL is the MIC that inhibits the growth of <i>E. coli,</i> whereas those for <i>S. aureus</i> are below the detection limit (<1.76 μg/mL). The tolerance limit of the mice model was inspected by injecting different concentration of Fe₂O₃ NPs and bacteria suspensions. The 14 ppm suspension was the tolerated dose and the concentration above were proved lethal. The most severe infection was induced in mice with injection of 3 × 10⁷ CFUs of both bacteria, while the inoculation of higher concentrations of bacterial suspensions resulted in the mice’s death. The histopathological and hematological studies reveals that the no/negligible infection was found in the mice exposed to the simultaneous inoculation of Fe₂O₃ NPs (14 ppm) and bacterial suspensions (3 × 10⁷ CFUs).