Barley (Hordeum vulgare L.) is one of the most used species for hydroponic green fodder. The chemical properties of fodders may be enhanced through use of various strategies during crop production, including stress applications. In this context, hydrogen peroxide (H₂O₂) is used as a stress factor in controlled elicitation, a technique used to increase secondary metabolites in food. The aim of this research was to evaluate the possibility of using controlled elicitation with H₂O₂ for enhancing the chemical properties of hydroponic barley fodder. H₂O₂ was applied to the seeds as a pre-germination treatment at three concentrations: 50, 100 and 150 mM. Morphological changes, enzymatic defence-related activities (superoxide dismutase, catalase, chalcone isomerase, phenylalanine ammonia-lyase), total phenolics content, and antioxidant capacity were evaluated. Significant increases in total phenolics content, phenylalanine ammonia-lyase activity and ABTS antioxidant capacity were obtained when seeds were treated with 50 mM H₂O₂. Plant growth was promoted with 100 mM and 150 mM H₂O₂ treatments, and germination of seeds with 100 mM H₂O₂. It was established that controlled elicitation using H₂O₂ increased some morphological and biochemical variables of hydroponic barley fodder related to food properties.