Submicron-sized iron oxide particles can influence the activity of bacteria, but the exact mechanisms of oxide toxicity toward bacteria remain elusive. By using atomic force microscopy (AFM), soft X-ray tomography (Nano-CT), and Fourier transform infrared (FTIR) spectrometry, we show how the size-dependent interfacial interactions between hematite particles and bacteria in the absence of any ligands contribute to the antimicrobial properties against Gram-positive and Gram-negative bacterial strains. We found that surface adhesion between hematite particles and bacterial cells is initially dominated by Lifshitz van der Waals and electrostatic forces. Subsequently, the rapid formation of P-O-Fe bonds occurs, followed by changes in the structures of membrane proteins in 2 h, resulting in the loss of the structural integrity of the membrane within 10 h. Thus, particles can migrate into the cells. After contact with bacterial cells, reactive oxygen species are generated on the surface of hematite particles, leading to cell permeabilization. G- bacteria appear to be more susceptible to this process than G+ bacteria because the latter exhibit weaker adhesion forces toward hematite and benefit from the protective effects of the peptidoglycan layers. Our work revealed that hematite nanoparticles are more toxic to bacteria than microscaled particles due to their strong interfacial physicochemical interactions with the cells.