While focus in oil-producing microalgae is normally on nutrient deficiency, we addressed the seasonal variations of lipid content and composition in large-scale cultivation. Lipid content, fatty acid profiles and mono- di- and triglycerides (MAGs, DAGs, and TAGs) were analyzed during May 2007–May 2009 in <em>Nannochloropsis oculata</em> grown outdoors in closed vertical flat panels photobioreactors. Total lipids (TL) ranged from 11% of dry weight (DW) in winter to 30% of DW in autumn. 50% of the variation in TL could be explained by light and temperature. As the highest lipid content was recorded during autumn indicating an optimal, non-linear, response to light and temperature we hypothesize that enhanced thylakoid stacking under reduced light conditions resulted in more structural lipids, concomitantly with the increase in glycerides due to released photo-oxidative stress. The relative amount of monounsaturated fatty acids (MUFA) increased during autumn. This suggested a synthesis, either of structural fatty acids as MUFA, or a relative increase of C16:1 incorporated into TAGs and DAGs. Our results emphasize the significant role of environmental conditions governing lipid content and composition in microalgae that have to be considered for correct estimation of algal oil yields in biodiesel production.