Zinc is considered one of the essential micronutrients for plants and, although required in small amounts, plays a significant role in plant growth and development. This element is particularly involved in the stimulation of flowering and the progression of plants toward maturity. However, excessive zinc accumulation may lead to toxicity and impair plant performance. Given the nutritional importance of spinach in the human diet and the widespread application of zinc oxide in various industries, the present study investigated the effects of zinc oxide application on growth, biochemical, and developmental traits of spinach (Spinacia oleracea L.) at the flowering stage. The results indicated that zinc oxide application improved several growth parameters in many treatments, including increases in shoot height, root length, leaf area, and fresh and dry biomass of plant organs. Biochemical analyses revealed treatment-dependent alterations in photosynthetic pigments, such as chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids, with increases observed in some treatments and decreases in others. The contents of flavonoids and phenolic compounds increased at lower zinc oxide concentrations but declined at higher levels. Moreover, zinc oxide treatment resulted in an overall increase in total protein content compared with control plants, particularly at lower concentrations. These findings suggest that the balanced application of zinc oxide can enhance growth and biochemical attributes of spinach, whereas excessive application may exert adverse effects.