► Neochloris oleoabundans lipids were analyzed as biodiesel feedstock. ► Cultures were gown in enriched seawater under optimal and N-stress conditions. ► Lipid accumulation was mainly due to a significant increase of triacylglycerols. ► Monounsaturated fatty acids were dominant and rich in oleic acid. ► Oil quality has shown a good performance for biodiesel production. The freshwater microalga Neochloris oleoabundans was used to study algal lipid production in enriched natural seawater, in order to assess its suitability as biodiesel feedstock. Optimal and nitrogen-stress (N-stress) conditions were analyzed. Under optimal conditions, the strain’s growth rate was 0.73 div day−1 and the biomass concentration was 1.5gL−1, while it had a maximum lipid yield under N-stress conditions (lipid content: 26% of dry weigh and lipid productivity: 56mgL−1 day−1). Lipid accumulation was mainly due to a significant increase of triacylglycerol content. Neutral lipids were characterized by a dominance of monounsaturated fatty acids and displayed a fatty acid profile that is suitable for biodiesel. This work offers an interesting alternative for sustainable microalgal oil synthesis for biodiesel production without using freshwater resources. However, further studies are necessary in order to optimize the lipid productivities required for commercial biodiesel production.