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• Effects of nitrogen on nutrient removal and biodiesel production of mono- and mix-cultured microalgae was investigated. • Microalgae co-culture could remove 70.4%–74.1% nitrogen. • Algae grown in media with different nitrogen sources had better growth performances. • The ultimate biomass increase and productivity in the mix-culture were the highest. • The biodiesel yields in this study ranged from 8.5–11.2 g-biodiesel/100 g-dry weight. Effects of nitrogen source inhomogeneity on nutrient removal and biodiesel production of mono- and mix-cultured microalgae were investigated in this study. The microalgal growth characteristics, lipid accumulation, biodiesel production and nutrient removal were determined by mono- or co-culturing microalgae Chlorella vulgaris and Scenedesmus dimorphus in media with different nitrogen sources. The results showed that microalgae cultivated in media with different nitrogen sources indicated higher nitrogen removal, biomass increase and productivities of biomass, lipids and biodiesel than media with the sole nitrogen source. As for the microalgae grown in media with the same nitrogen source, the pattern for the lipid content was in the relationship C. vulgaris > mixed culture > S. dimorphus. The fatty acids of microalgae mainly consisted of C16:0, C16:1, C18:0, C18:2 and C18:3, accounting for 79.6–90.6% of the total. The biodiesel production in this study ranged from 8.5 to 11.2 g-biodiesel/100 g-dry weight. This study presents one of the few studies, suggesting that nitrogen source itself can influence the nutrient removal and biodiesel production of mono- and mix-cultured microalgae. [ABSTRACT FROM AUTHOR]