Abstract The high costs and environmental concerns associated with using marine resources as sources of oils rich in polyunsaturated fatty acids have prompted searches for alternative sources of such oils. Some microorganisms, among them members of the genus Aurantiochytrium, can synthesize large amounts of these biocompounds. However, various parameters that affect the polyunsaturated fatty acids production of these organisms, such as the carbon and nitrogen sources supplied during their cultivation, require further elucidation. The objective of this investigation was to study the effect of different concentrations of carbon and total nitrogen on the production of polyunsaturated fatty acids, particularly docosahexaenoic acid, by Aurantiochytrium sp. ATCC PRA-276. We performed batch system experiments using an initial glucose concentration of 30 g/L and three different concentrations of total nitrogen, including 3.0, 0.44, and 0.22 g/L, and fed-batch system experiments in which 0.14 g/L of glucose and 0.0014 g/L of total nitrogen were supplied hourly. To assess the effects of these different treatments, we determined the biomass, glucose, total nitrogen and polyunsaturated fatty acids concentration. The maximum cell concentration (23.9 g/L) was obtained after 96 h of cultivation in the batch system using initial concentrations of 0.22 g/L total nitrogen and 30 g/L glucose. Under these conditions, we observed the highest level of polyunsaturated fatty acids production (3.6 g/L), with docosahexaenoic acid and docosapentaenoic acid ω6 concentrations reaching 2.54 and 0.80 g/L, respectively.