Chalcone is a secondary metabolite belonging to the group of flavonoids. It has shown strong phytotoxic activity on Arabidopsis roots, as inductor of programmed cell death, and inhibitor of root growth and root hair formation. Peroxidases are particularly abundant in root meristems and are involved in the formation and interconversion of reactive oxygen species (ROS), which play a critical role on root and root hair development. Therefore, we report here the role of peroxidases in Arabidopsis root development during chalcone treatment. A strong inhibition of peroxidase activity was detected in the apical root meristems after chalcone treatment, which reflects the important role of these enzymes on the mode of action of this secondary metabolite.