Abstract: To investigate the effects of nitrogen on the growth and lipids accumulation of Eustgmatophyceae, three different nitrogen sources (sodium nitrate, ammonium bicarbonate and urea) and four different initial nitrogen concentrations (18 mmol, 9 mmol, 6 mmol and 3 mmol) were used to culture eight species of Eustgmatophyceae (Eustigmatos cf. polyphem, Eustigmatos magnus, Eustigmatos polyphem, Eustigmatos vischeri, Vischeria helvetica, Vischeria punctata, Vischeria stellata and Nannochloropsis oculata) to select the best culture condition. The pH values of culture medium, biomass, lipids accumulation and fatty acid composition of Eustgmatophyceae were utilized to assess its effects. The results showed that three kinds of nitrogen sources were suitable for the eight species. For the prolong culture, the pH values of culture medium gradually reduced to 5.0—6.0 when ammonium bicarbonate and urea were used as nitrogen sources, and the pH values of culture medium remained at 7.0—8.0 when sodium nitrate was used as nitrogen sources. Meanwhile, the higher biomass could be obtained when urea was used as nitrogen source, but different species reached to the highest biomass under different nitrogen concentrations. The highest biomass of 10.96 g/L was achieved in E. Polyphem at 9 mmol. Low nitrogen level could stimulate the lipids accumulation in all species with the highest lipid content of 59.24% of dry weight in E. vischeri. The total fatty acid contents of the eight species were about 50%—58% of dry weight, and the mainly fatty acids were myristic acid (C14鲶0), palmic acid (C16鲶0), palmitoleic acid (C16鲶1), oleic acid (C18鲶1) and eicosapentaenoic (C20鲶5). In N. oculata, the content of palmic acid was the highest one, which was about 50% of total fatty acids. However, in other seven species, the content of palmitoleic acid reached about 40%—60% of total fatty acids. In conclusion, urea (at 6 mmol nitrogen concentrations) is the best condition for the eustigmatophycean microalgae to accumulate biomass and oil. In general, all eight species of Eustgmatophyceae can accumulate large contents of lipids, and Eustigmatos showed the best potential to produce biofuels.