A hydroponic experiment was conducted to explore the interactive effects of selenium (Se) supplementation (0, 5, and 10 μM) and arsenic (As) toxicity (0, 200, and 400 μM) on the growth, accumulation, and oxidative damage along with defense mechanisms of metallicolous (MP) and non-metallicolous population (NMP) of Isatis cappadocica, an As-hyperaccumulator, and Brassica oleracea as reference brassica. The results revealed that As stress significantly hampered plant growth particularly in B. oleracea. It reduced plant growth due to enhanced oxidative load of As-stressed plants. Between the two Isatis populations, metallicolous plants accumulated significantly higher As, however with considerably low growth defects. Furthermore, Se supplementation counteracted the adverse effects of stress on growth and physiological performance of all studied plants. Addition of Se, particularly at higher dose (10 μM), significantly suppressed root As uptake and slightly its accumulation in shoots of B. oleracea plants treated with 400 μM As, and thus improved growth characteristics of stressed plants. Under As stress, Se supplementation increased the activities of enzymatic (peroxidase (POD) and glutathione reductase (GR)) and non-enzymatic (anthocyanins and total flavonoids) antioxidants, thereby suggesting relieved As stress by reduced oxidative damage. Taken together, these results support the beneficial role of Se in the regulation of As stress by improving growth, physiology, and antioxidant capacity, and highlight its significance for plants grown on such metal-contaminated soils.