Due to its nutritional value, wheatgrass (Triticum aestivum L.) is considered to be a functional food, becoming increasingly popular as a supplement to the people’s quotidian diet. The study aimed to determine the influence of the number of cuttings and cultivars on the total antioxidant activity (DPPH), the content of chloroplast pigments, vitamin C, phenols, and flavonoids in the wheatgrass juice. Two genotypes of wheatgrass, T. aestivum ssp. aestivum (variety Katarina) and T. aestivum ssp. sphaerococcum, respectively, were cut twice during the experiment. In both cuttings, the genotype significantly differed in the flavonoid level and antioxidant activity, while the number of cuttings influenced the content of phenols, vitamin C, and antioxidant activity in the wheatgrass juice. T. sphaerococcum had a higher concentration of flavonoids and a significantly lower antioxidant activity when compared to the Katarina wheat variety. On an average, the first cut implicated an increased content of phenols and vitamin C concerning both genotypes, followed by a higher antioxidant value. In the Katarina variety, a significantly higher phenol content and antioxidant activity was detected in the first cut. In the T. Sphaerococcum, a decrease in the total content of the examined antioxidants was apparent in the second cut.