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We synthesize four hematite nanomaterials of different sizes, shapes, and crystal structures. After characterizing the size, shape, crystallinity, and specific surface area of these materials through transmission electron microscopy (TEM), dynamic light scattering (DLS), x-ray diffraction (XRD), and the Brunauer–Emmett–Teller method (BET), we examine their toxicity towards E. coli MG1655. By modifying a standard colony forming unit (CFU) count toxicity assay to isolate the role of dissolved ions and reactive oxygen species (ROS), we elucidate the specific chemical and biological pathways responsible for toxicity. Toxicity is found to stem from both dissolved Fe2+ ions as well as ROS, the latter of which may be generated on contact between particles and cells and is produced in greater quantities under UV light. This investigation clarifies the mechanism of antimicrobial activity in hematite nanomaterials, and represents an important step towards design of materials for maximum functionality and minimum hazard.