The purpose of this study was to assess the effects of nitrogen (N) fertilization and cultivar on vitamin C content, total antioxidant capacity (TAC), and catalase (CAT) activity in amaranth leaves, as well as in the rhizosphere and non-rhizosphere soil. For this purpose, a 3-year split-plot field experiment was conducted, which included the following factors: N fertilization (kg ∙ ha–1: N0 – control, N1 – 60, N2 – 90, N3 – 120, N4 – 150), amaranth cultivars (‘Rawa’ and ‘Aztek’) and developmental stages (BBCH 13, BBCH 16, BBCH 19). The factor that most significantly differentiated the vitamin C content in the leaves was the cultivar, followed by the development stage, N fertilization, and weather conditions. The cultivar ‘Aztek’, stage BBCH 13, N3 fertilization, and weather conditions during the last year of the experiment gave the best results in this regard. CAT activity in the leaves significantly depended on N fertilization and developmental stage. It increased with the higher N dose and decreased with plant development. The TAC of the leaves depended only on the developmental stage and weather conditions. The highest TAC was observed at BBCH 13 and the third year of the study. CAT in the rhizosphere significantly depended on N fertilization, cultivar, and developmental stage, while in the non-rhizosphere zone, it depended on N fertilization, developmental stage, and weather conditions. This study is an essential addition to the knowledge on the use of amaranth seed forms as a vegetable with high nutritional value and antioxidant properties, as well as the effect of this plant on soil biological properties.