Abstract: To evaluate the impact of pH on the nutritional quality of dietary diatoms and determine the pH threshold at which changes in nutritional value occur, the responses of Skeletonema costatum and Thalassiosira weissflogii to pH variations were studied. The photosynthetic physiology and fatty acid composition of both the species were investigated by manipulating pH in the culturing system. Results show that, within the pH range of 7.0 to 8.0, pH had no significant effects on the growth, relative electron transfer rate, and fatty acid composition of both diatoms. However, when culturing pH increased to 9.0, the growth rate and effective quantum yield of photosystem II in both diatoms were moderately inhibited, with an increase in soluble protein and soluble carbohydrate in T. weissflogii. The saturated fatty acid content in S. costatum exceeded 90% of the total content of fatty acid content in all treatment groups, with the highest content of docosahexaenooic acid (DHA) at pH 9.0. The synthesis of unsaturated fatty acids in T. weissflogii was promoted in the pH range of 7.0 to 8.0, with the highest DHA and eicosapentaenoic acid content observed at pH 7.0. These findings offer important insights for cultivation guidance and improving the nutritional quality of dietary microalgae.