The microalga Scenedesmus obliquus was cultured in two cultivation stages: (1) in batch with real wastewater; (2) maintaining the stationary phase with different conditions of CO2, light and salinity according to a factorial design in order to improve the lipid content. The presence of the three factors increased lipid content from 35.8% to 49% at the end of the second stage; CO2 presence presented the highest direct effect increasing lipid content followed by light presence and salt presence. The ω-3 fatty acids content increased with CO2 and light presence acting in isolation, nevertheless, when both factors acted together the interaction effect was negative. The ω-3 eicosapentaenoic acid content of the oil from S. obliquus slightly exceeded the 1% maximum to be used as biodiesel source (EU normative). Therefore, it is suggested the blend with other oils or the selective extraction of the ω-3 fatty acids from S. obliquus oil.