Melatonin, an important phytochemical, encourages plants to redirect growth in response to environmental stresses. The study aims to investigate the changes in the morphological, physiological, biochemical, and molecular properties of spinach subjected to the exogenous application of melatonin (MEL) at different doses under different water stress. The following four irrigation levels were applied: I100 (complete irrigation), I80 (20% water deficit), I60 (40% water deficit), and I40 (60% water deficit). MEL was applied through a spray on the leaves in three doses—0, 50, and 100 µM. In the present study, the application of MEL50 exhibited no significant decrease in water use efficiency (WUE) at the I80 irrigation level compared to the I100 irrigation level, while a significant decrease in the WUE was observed beyond this point. The application of MEL contributed to influencing the morphological parameters while also positively affecting the photosynthesis activity and, contribution to the antioxidant defense system. It was observed that the genes involved in the carbon metabolism of photosynthesis), the antioxidant mechanism, and the continuity of photosynthesis, all of which affect the expression of melatonin, facilitated water stress reduction in spinach. Therefore, it was inferred that the application of MEL50I80 could serve as an important irrigation strategy in semi-arid regions with limited water resources.