Different sources of nitrogen pose diverse effects to algal community, but the mechanism of inhibitory effects of nitrogen sources on freshwater diatoms is not fully understood. The purpose of this study was to compare biomass, photosynthetic activity, and morphological structure of three common freshwater diatoms (Cyclotella meneghiniana, Nitzschia sp., and Gomphonema parvulum) under different nitrogen sources (NO3 − or NH4 +). The sorption characteristic of each diatom was investigated, and chlorophyll a (Chl-a) content and oxygen evolution rate were analyzed to investigate stress of different nitrogen sources on each diatom in the batch experiments. Ammonium lowered the growth rate of C. meneghiniana and Nitzschia sp. when it was supplied in addition to growth-saturating nitrate concentrations, suggesting a combined effect of inhibition of nitrate uptake and direct ammonium stress. Oxygen evolution rate of Nitzschia sp. showed that the direct ammonium stress on the photosynthetic activity can be alleviated by coexistence of nitrate in the nitrogen enriched treatment, but not for C. meneghiniana and G. parvulum, which may be caused by a different nitrate transporter system within algal cells. Transmission electron microscopy was used to assess the toxicity of ammonium on ultrastructural chloroplast of each diatom. Ultrastructural changes in chloroplasts showed undefined electron-dense granules and lipid droplets, but the membrane integrity of cell was maintained, suggesting an adaptation to adjustment to ammonia stress. Results showed that Cyclotella meneghiniana and Nitzschia sp. were more sensitive to ammonium stress than Gomphonema parvulum on growth, but the mechanism remains unclear.