Microalgae can accumulate lipids during the stationary growth phase, but little information is available about fatty acid profile changes during this phase to determine the best harvesting time in respect to lipid content. In this study, Dunaliella sp. was cultured in F/2 medium at three different photon flux densities (380, 226 and 8.2 μmol photon m-2 s-1) and three different glycerol concentrations (0, 10 and 20 g L-1). Samples were taken during the stationary phase to assess lipid content and fatty acid profile variations. Microalgal biomass production was higher at 380 and 226 μmol photon m-2 s-1 than at 8.2 μmol photon m-2 s-1 in accord to light limitation. The maximum lipid content (345.78 mg g-1) was achieved at 8.2 μmol photon m-2 s-1 and 20 g L-1 glycerol at day 12, similar to that achieved at day 9 (334.16 mg g-1). The maximum polyunsaturated fatty acid amount (65.30 μg mg-1) was achieved at day 7 of culture without glycerol addition, decreasing in proportion over time. So, the best conditions and harvesting time in respect to fatty acid quality would be at 380 μmol photon m-2 s-1 without glycerol addition and after 7 days of culture.