The Paeonia emodi (P. emodi)-mediated iron oxide nanoparticles (Fe₂O₃ NPs) were screened for in-vitro and in-vivo antibacterial activity against the Staphylococcus aureus (S. aureus) (ATCC #: 6538) and Escherichia coli (E. coli) (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 E. coli, whereas those for S. aureus 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).