The genotoxin colibactin is a secondary metabolite produced by a variety of pathogenic Enterobacteria, and is associated with colon cancer development and acute systemic infections. The colibactin biosynthesis requires the enzymatic activity of the phosphopantetheinyl transferase ClbA. We recently evidenced that two master regulators of bacterial iron homeostasis, i.e. the ferric uptake regulator (Fur) and the small regulatory non-coding RNA RyhB, were involved in the regulation of the clbA transcription and of the colibactin production. In this study, we investigated the impact of high iron supply on clbA transcription and colibactin production in wild type, ΔryhB, Δfur and ΔryhB Δfur strains. This revealed that high iron resulted in decreased synthesis of the genotoxin colibactin through both pathways dependent and independent of Fur/RyhB. This work highlights the complex regulatory mechanism that controls an important bacterial virulence and carcinogenesis factor by regulators of bacterial iron homeostasis.