Nitrogen fertilization may affect the functioning of photosynthesis as well as the chemical composition and antioxidant potential of cereal grains. Little is known about the relationship between the efficiency of photosynthesis and the content of phenolic compounds in barley grain, especially in conditions of varying nitrogen availability. In this regard, a field experiment was conducted to examine the responses of two primary barley genotypes with elevated phenolic compound content (TPC) in grain and an intensive modern cultivar H. v. vulgare with high protein content to increasing nitrogen fertilization (rates of 0, 30, 60 and 90 kg N ha−1) during the study years, which differed in terms of hydrothermal conditions. The leaf greenness index (SPAD) and chlorophyll fluorescence parameters were evaluated on three occasions throughout the growing season. Following the harvest, the chemical composition of the grains, including phenolic acids, flavonoids and antioxidant potential, was evaluated. The antioxidant potential and chemical composition of the grain, including TPC and protein content, depended to the greatest extent on genetic and environmental factors, and only then on nitrogen fertilization. Nitrogen increased the TPC content and antioxidant capacity ABTS+ of the grains of all studied genotypes and the protein content in H. v. vulgare grain. Rates of 60 and 90 kg N ha−1 resulted in a significant increase in the SPAD, PIabs and Fv/Fm in BBCH 34 and 57. A positive correlation was confirmed between the SPAD and PIabs and the content of TPC and ABTS+ in the grain. The dependence of qualitative characteristics on the Fv/Fm was also demonstrated. The primary genotypes are characterized by a greater genetic potential for the synthesis of phenolic compounds than the modern cultivar H. v. vulgare. The synthesis of phenolic compounds, and thus their accumulation in the grain, is clearly stimulated by unfavorable environmental factors and moderate nitrogen rates and depends on the chlorophyll content in the leaves and the efficiency of photosynthesis. N fertilization has a beneficial effect on the content of phenolic compounds in grain resulting from the improvement in the SPAD and PIabs. The chemical composition of grain and the increase in antioxidant potential are determined by the Fv/Fm, which is low under hydrothermal stress conditions.