The aim of this study was to evaluate the impact of combined biofortification of spinach with iodine (I) and selenium (Se). For this purpose, the spinach plant was cultivated in an open field (Dehdasht, Iran), biofortified with potassium iodide (KI) and sodium selenate (Na₂SeO₄) at different concentrations (Se 1 mg/L-I 1 mg/l; Se1-I1, Se2.5-I1, Se5-I1, Se1-I5, Se2.5-I5, and Se5-I5) through spraying the leaves twice during the growth season. Results indicated that while iodine did not have any effect on plant yield, selenium fortification at 2.5 mg/L significantly increased production (60.05 t/ha). However, both elements were successfully accumulated in the leaves of the plant. Therefore, the highest accumulation for both elements was noted by Se5-I5 sample. Meanwhile, the joint biofortification of spinach improved the activity of antioxidant enzymes, macro/microelements content, photosynthetic pigments, nitrate reductase activity, ascorbic acid, total phenol content, carotenoid compounds, TSS, and dry matter percentage, while decreasing the nitrate and malondialdehyde contents in the leaves, resulting in a plant with improved dietary properties and yield production. In this regard, treatment Se2.5-I5 was the best treatment in relation to various tests conducted.