Global warming and the subsequent climate change increase the severity and frequency of floods day by day. Therefore, there is a need to develop new strategies to prevent the negative effects of flooding stress and to determine the changes that may occur in the plant under extreme water stress. Thus, in this study, changes in agronomic, physiological, and biochemical properties of lettuce plants exposed to flooding stress under greenhouse conditions were analyzed. In this study, two different irrigation regimes, including full irrigation (I100) and flooding stress (I150) and three different nitrogen doses, including no-nitrogen (N0), 100 kg ha−1 (N10), and 200 kg ha−1 (N20), were applied. The results revealed that while flooding stress caused significant decreases in above-ground fresh and dry weights, membrane damage (MD), contents of carotenoid (CT), proline (PL), and protein (PT), as well as catalase activity (CAT), leaf relative water content (RWC) and peroxidase (POX) content showed significant increases. Increasing nitrogen doses caused the increase in the pigment contents (Cl a, Cl b, and CT) and PL, superoxide dismutase (SOD) of the lettuce. In addition, RWC content and the uptake of Fe and Mn elements increased under flooded conditions. It has been revealed that the application of at least 100 kg of nitrogen fertilizers per hectare to prevent the negative effects of flooding stress in the regions where lettuce cultivation was carried out supported the plant growth both under full irrigation and flooded conditions.