Abstract To assess the effect of supercritical fluid CO2 (SCFCO2) extraction on the biochemical composition of algal biomass, samples of untreated Nannochloropsis granulata biomass (Initial) and residual biomass after SCFCO2 extraction at 70 and 90 °C (C70 and C90) were analyzed. SCFCO2 extraction significantly reduced crude lipid content from 285.5 g/kg to 256.2–256.3 g/kg (P=0.032) and caloric content from 23.4 MJ/kg to 23.0–23.1 MJ/kg (P=0.004). Concomitantly, significant increases in ash content from 77.8 g/kg to 83.8–86.2 g/kg (P=0.002) and carbohydrate content from 149.1 g/kg to 155.9–165.9 g/kg (P=0.033) were observed and no significant differences between C70 and C90. Small, but significantly lower levels of crude protein (CP, N × 6.25 or N × 4.78) and non-protein N (NPN) were observed for C70 with no differences between Initial and C90. However, sum of amino acid (AA) residues (ΣAA), a more direct estimate of true protein content, indicated no significant difference in protein content (P=0.536) between treatments (average, 362.0 g/kg). Only minor differences in total and free AA profiles were observed between Initial, C70 and C90 and the free AA content as percentage of total AA was insignificant (P=0.564) between treatments (average, 4.9%). However, several free essential AAs (EAAs) were significantly higher in C70 and C90 than Initial resulting in SCFCO2-extracted biomass having significantly higher (P=0.007) total free EAA content (2438.0–2697.0 μg/g of DW) than Initial (2100.4 μg/g of DW) indicating some protein damage and free EAA liberation. While there was a trend toward most AAs slightly increasing in C70 and C90, the EAA lysine; particularly sensitive to high-temperature processing, was significantly lower (P=0.003) in C90 (6.0 g lysine/100 g protein) than Initial (6.9 g lysine/100 g protein) while C70 was intermediate (6.4 g lysine/100 g protein) providing evidence that lysine damage was more severe at 90 °C than 70 °C. The essential AA index (EAAI) of 0.9 for N. granulata biomass is highly comparable to reference proteins such as egg albumin, soy, Chlorella and Spirulina (0.9–1.0) indicating very good potential for use in animal and fish feeds. Further investigations involving species-specific in vitro protein quality and in vivo biological performance of target animals fed diets supplemented with these products are warranted.