We investigated the potential use of various carbon sources (fructose, glucose, mannose, lactose, and glycerol) for culturing Phaeodactylum tricornutum UTEX-640 in mixotrophic and heterotrophic batch cultures. Concentrations of carbon substrates tested ranged from 0.005 M to 0.2 M. P. tricornutum did not grow heterotrophically on any of the C-sources used, but successive additions of organic carbon in mixotrophic growth mode substantially increased the biomass concentration and productivity relative to photoautotrophic controls. The maximum biomass productivities in mixotrophic cultures for glycerol, fructose, and glucose were 21.30 mg/l h, 15.80 mg/l h, and 10.20 mg/l h, respectively. These values were respectively 10-, 8-, and 5-fold higher than those obtained in the corresponding photoautotrophic control cultures. Mannose and lactose did not significantly affect microalgal growth. The biomass lipids, eicosapentaenoic acid (EPA) and pigments contents were considerably enhanced with glycerol and fructose in relation to photoautotrophic controls. The EPA content was barely affected by the sugars, but were more than 2-fold higher in glycerol-fed cultures than in photoautotrophic controls.