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Concerns about greenhouse gas emissions and climate change have raised global demand for renewable products, leading to increased interest in sustainable resources. Microalgae constitute a potentially sustainable source of natural compounds, such as phospholipids, which are extensively used in cosmetics as part of liposomes, emulsifiers, solubilizers, and wetting agents. Currently phospholipids are extracted from food sources, a practice that raises sustainability concerns. Photosynthetic microalgae cultivated on nitrogen and phosphorus macronutrients may serve as a more sustainable source of the major phospholipids phosphatidylcholine, phosphatidylethanolamine, lyso-phosphatidylcholine, and lyso-ethanolamine. We assessed the effect of nitrogen and phosphorous provision on phospholipid production by the marine microalga Nannochloropsis oculata cultivated in a 3.5-liter flat panel photobioreactor at two macronutrient mass ratios, NO3-/PO43- = 15 and NO3-/PO43- = 5. Using organic solvent extraction and P-31 NMR spectroscopy, phospholipid content in cell mass was found to be 26% higher at the ratio of 5 than at the ratio of 15, indicating that phosphate-rich cultivation boosts phospholipid formation. Moreover, the specific growth rate of the culture at the ratio of 5 was higher than at the ratio of 15. After the growth phase the cultures were subjected to macronutrient starvation, a common practice for increasing total lipid production. Therefore, maintaining a NO3-/PO43- ratio of 5 is deemed to be essential for exploiting microalgae for large scale production of phospholipids.