► Nannochloropsis sp. was studied as a biorefinery context. ► Production of fatty acids for biodiesel, high added-value compounds and biohydrogen. ► Extracts fractionation by SFE (oils for biodiesel and pigments for food). ► Fermentation of biomass leftover by E. aerogenes yielded maximum 60.6mLH2/gdry biomass alga. ► Best SFE conditions extracted 45glipids/100gdry biomass (40°C, 300bar, CO2+ethanol). The microalga Nannochloropsis sp. was used in this study, in a biorefinery context, as biomass feedstock for the production of fatty acids for biodiesel, biohydrogen and high added-value compounds. The microalgal biomass, which has a high lipid and pigment content (mainly carotenoids), was submitted to supercritical CO2 extraction. The temperature, pressure and solvent flow-rate were evaluated to check their effect on the extraction yield. The best operational conditions to extract 33glipids/100gdrybiomass were found to be at 40°C, 300bar and a CO2 flow-rate of 0.62g/min. The effect of adding a co-solvent (ethanol) was also studied. When supercritical CO2 doped with 20% (w/w) ethanol was used, it was possible to extract 45glipids/100gdrybiomass of lipids and recover 70% of the pigments. Furthermore, the remaining biomass after extraction was effectively used as feedstock to produce biohydrogen through dark fermentation by Enterobacter aerogenes resulting in a hydrogen production yield of 60.6mL/gdrybiomass.