Global climate change is increasing its negative impact on agricultural production day by day. Abiotic stress factors brought about by climate change cause significant losses in yield, quality, and plant growth. In addition to increasing the effects of drought and salinity with global warming, changes in precipitation regimes and flooding stresses have recently negatively affected agricultural areas. To avoid the negative effects of stress, proline (Pro) and glycine betaine (GB) applications are the subject of research as important applications. This study, it was aimed to determine the effects of Pro and GB doses on growth, physiological and biochemical parameters applied under conditions artificially created ten-day flooding stress in green onions. One full irrigation subject (I100) and one stress subject (flooding), were created in the experiment. On the other hand, three different Pro doses (Pro1–1 μM, Pro2–2 μM, and Pro3–3 μM) and three different GB doses (GB50–50 μM, GB100–100 μM, and GB150–150 μM) administration and control were the other subjects of the study. Pro and GB applications were applied to the leaves as a spray twice. As a result of the study, flooding stress revealed 28% loss in leaf number, 43% plant fresh weight, and 32% plant dry weight loss. Malondialdehyde (MDA), hydrogen peroxidase (H2O2), and leaf temperature (T leaf) flooding stress conditions were found to be the parameters that provide the most significant increase and define the stress. It was observed that Pro2, Pro3, and GB50 applications supported plant growth under full irrigation conditions. Besides, it was revealed that Pro3 and GB50 doses reduced the negative effects of flooding stress. It has been concluded that appropriate doses of Pro and GB applications in terms of sustainable agriculture are important applications in reducing the negative effects of stress.