Aim The aim of this study was to test the hypothesis that Manuka honey (MH) chelates iron and promotes an iron-limiting environment, which contributes to its antimicrobial activity. Methods and Results Employing a ferrozine-based assay, we observed that MH is an iron chelator that depletes iron from solution. Siderophores are small molecules that bind ferric iron (III) with high affinity and their levels are upregulated by bacteria under iron-limiting conditions. We demonstrated by quantitating siderophore production that Escherichia coli and Pseudomonas aeruginosa treated with MH sub-minimum inhibitory concentrations (sub-MIC) experience an iron-limiting environment and increase siderophore production. In addition, supplementation with ferrous iron (II) significantly increased growth of E. coli, Staphylococcus aureus and P. aeruginosa cultured at their MH MIC above that observed in nonsupplemented controls. By contrast, supplementation with ferric iron (III) significantly increased growth for only E. coli and P. aeruginosa, above their nonsupplemented controls. Conclusions Manuka honey chelates iron, thereby generating an iron-limiting environment for E. coli and P. aeruginosa, and to a lesser extent S. aureus, which contributes to its antimicrobial properties. Significance and Impact of the Study Our work demonstrates that MH-induced iron chelation is an antimicrobial mechanism that differentially impacts the bacterial species tested here. Iron chelation affects multiple diverse physiological processes in bacteria and would contribute to the lack of bacterial resistance to MH. Iron metabolism is tightly regulated; bacteria require this essential nutrient for survival, but in excess it is toxic. Additional exploration of MH's iron chelation mechanism will facilitate its future use in mainstream medicine.