Despite being a potential source of bioactive products with antioxidant properties, microalgae are not yet directly consumed as traditional food. Currently only certain extracted compounds are used as ingredients and supplements. Nannochloropsis gaditana is one of the most promising microalgae as source of omega-3, polyunsaturated fatty acids (PUFA’s) whose intake is necessary in the diet against cardiovascular problems. The extraction of EPA-rich oils derived from microalgae still needs to be experimented with supercritical carbon dioxide extraction (CO2-SFE) to increase extraction yields and maintain nutritional properties. CO2-SFE is an ideal technology to achieve this goal for mild operational conditions and the selectivity of CO2 in supercritical condition with non-polar compounds such as lipids. Moreover, there is still a very limited knowledge available at pilot-scale studies level and not much work has been carried out on the process scale up. Therefore, the present work was carried out with the objective to test the CO2-SFE pilot scale extraction from the microalga N. gaditana, to compare the results with bench scale studies and to study the effect of biomass stirring during extraction. The results showed a positive effect of stirring on lipids and EPA recovery compared to the session without stirring. The extraction yields achieved at pilot scale were resulted higher comparable to those obtained on a bench scale in previous work. © 2023 ETA-Florence Renewable Energies.