Flooding stress is an important abiotic stress factor, restricting agricultural production and causing yield and quality losses. This study was carried out to measure the effects of melatonin (MEL) applications on plant growth and the physiological and biochemical changes associated with flooding stress in seedling cauliflower plants. Approximately one-month-old cauliflower seedlings were subjected to flooding stress (Flo) and compared with fully irrigated control plants. Three doses (0, 50, and 100 µM) of MEL were applied to the seedlings in the form of a spray, and the plants were harvested 10, 15, and 20 days after Flo and MEL treatments. Agronomic, physiological, and biochemical measurements were made from the plant samples taken after each harvest. The Flo applied to the cauliflower seedlings significantly restricted their growth, causing reductions in aboveground fresh and dry weight, under-ground fresh and dry weight, and leaf area of 68%, 58%, 45%, 62%, and 40%, respectively. Principal component analysis (PCA) revealed that the most important factor of the study was Flo. Plant growth parameters, leaf relative water content, chlorophyll b, stomatal conductance, actual photosynthetic efficiency, and chlorophyll fluorescence values gave the best results under full irrigation conditions; while under Flo conditions, malondialdehyde, peroxidase, hydrogen peroxidase, chlorophyll a, carotenoid, catalase, and leaf temperature exhibited significant changes. The MEL treatments had little effect in ameliorating stress conditions. In conclusion, the exposure of agricultural lands to flood stress is an important consideration when determining where cauliflower will be grown.