<p>Exposure to chronic cold stress has been shown to affect the performance of Nile tilapia, resulting in substantial economic losses during the winter and early spring months. Implementing appropriate and economically viable nutritional strategies has proven to be an effective measure to maintain welfare and ensure satisfactory performance under low-temperature conditions. We evaluated the effects of dietary supplementation with prebiotics mannanoligosaccharides (MOS), β-glucans (BG), and their combination (MOS + BG) on the innate immune response and intestinal morphology of Nile tilapia subjected to prolonged cold stress. Prebiotic supplements significantly improved innate immune function, intestinal health, and some hematological parameters such as neutrophil/lymphocyte ratio, although they did not significantly affect growth performance or blood biochemistry (<i>p</i> &gt; 0.05). The concentration of lysozyme in serum was increased by MOS (<i>p</i> &lt; 0.01), and lysozyme in mucus was increased by MOS and MOS + BG (<i>p</i> &lt; 0.001) compared to the control diet. The combined diet (MOS + BG) increased the respiratory activity of leukocytes (<i>p</i> &lt; 0.01) compared to the diet without prebiotics. Leukocyte phagocytic function and intestinal health were higher (<i>p</i> &lt; 0.01) in fish that received prebiotic supplements than in those that received the control diet. MOS supplementation, alone or in combination with β-glucans, was more effective in stimulating innate immunity and mitigating the adverse effects of cold stress. In turn, supplementation with prebiotics generally contributed to preserving the intestinal integrity of the fish under hypothermic conditions. Strategic administration of prebiotics derived from the yeast <i>Saccharomyces cerevisiae</i> in winter diets may help reduce cold-related mortality and improve economic outcomes in tilapia farming.</p> Graphical Abstract <p></p>