In this biosafety risk assessment project, the molecular and physiological responses of chicory seedlings to the introduction of selenate (0, 0.1, 0.5, 1, and 5 mgl-1) or nanoscale red elemental Se product (nSe) into the culture medium were investigated. The application of nSe at low concentrations improved the fresh weight of shoots and roots, while 5 mgl-1 nSe caused severe phytotoxicity. Molecular analysis confirmed partially different epigenetic responses to nSe and selenate. DNA hypomethylation is an important mechanism by which Se exerts its influence at the pre-transcriptional level. With increasing nSe concentration, the transcription factor DREB1A (dehydration-responsive element-binding) showed a linear upward trend. The use of nSe contributed to the transcriptional upregulation of the genes for phenylalanine ammonia-lyase (PAL), hydroxycinnamoyl-CoA: quinate-hydroxycinnamoyl transferases (HQT) and hydroxycinnamoyl-CoA: shikimate/quinate-hydroxycinnamoyl transferase (HCT). Proline concentrations were increased in both leaves and roots in response to the nano-supplement. Cytotoxicity of Se at toxic concentrations decreased protein levels, in contrast to the positive nSe treatments, 0.1 and 0.5. Notably, nSe supplementation acted as an efficient elicitor, stimulating the accumulation of phenylpropanoid derivatives, including caffeic acid, chlorogenic acid, and cichoric acid metabolites. The concentration of ascorbate and glutathione displayed a similar upward trend in response to the nSe supplementation. Further comprehensive comparative molecular studies in different stress-sensitive and tolerant species are necessary to gain a better understanding of the underlying mechanisms. This will allow for the optimization of functional protocols for nSe-based supplements to meet the expectations of sustainable agriculture.