Sustainable agricultural practices are still essential due to soil degradation and crop losses. Recently, the relationship between plants and nanoparticles (NPs) attracted scientists’ attention, especially for applications in agricultural production as nanonutrition. Therefore, the present research was carried out to investigate the effect of Fe3O4 NPs at low concentrations (0, 1, 10, and 20 mg/L) on three genotypes of barley (Hordeum vulgare L.) seedlings grown in hydroponic conditions. Significant increases in seedling growth, enhanced chlorophyll quality and quantity, and two miRNA expression levels were observed. Additionally, increased genotoxicity was observed in seedlings grown with NPs. Generally, Fe3O4 NPs at low concentrations could be successfully used as nanonutrition for increasing barley photosynthetic efficiency with consequently enhanced yield. These results are important for a better understanding of the potential impact of Fe3O4 NPs at low concentrations in agricultural crops and the potential of these NPs as nanonutrition for barley growth and yield enhancement. Future studies are needed to investigate the effect of these NPs on the expression of resistance-related genes and chlorophyll synthesis-related gene expression in treated barley seedlings.