Barley (Hordeum vulgare) grain stands out among other cereals due to its high nutritional value. It results mainly from the high content of fiber and antioxidants, such as phenolic compounds. Barley grains can also be an important source of unsaturated fatty acids and carotenoids that are beneficial to health. This study assessed the effect of the foliar application of an amino-acid biostimulant on the content of phenolic compounds, carotenoids, and the composition of fatty acids in the grain of alternative, black-grain barley genotypes, and the commonly used ‘Soldo’ cultivar, grown in conventional and organic farming systems. The dark-pigmented grains contained significantly more phenolic acids and flavonoids than the yellow seed of the traditional cultivar and were characterized by a significantly higher proportion of unsaturated fatty acids. The application of the biostimulant significantly increased the concentration of phenolic compounds in grains, especially of alternative genotypes.