Microalgae Dunaliella salina contains useful molecules such as β-carotene and fatty acids (FAs), which are considered high value-added compounds. To extract these molecules, supercritical carbon dioxide was used at different operative conditions. The effects of mechanical pre-treatment (grinding speed at 0⁻600 rpm; pre-treatment time of 2.5⁻7.5 min) and operating parameters for extraction, such as biomass loading (2.45 and 7.53 g), pressure (100⁻550 bars), temperature (50⁻75 °C) and CO₂ flow rate (7.24 and 14.48 g/min) by varying the extraction times (30⁻110 min) were evaluated. Results showed that the maximum cumulative recovery (25.48%) of β-carotene was achieved at 400 bars and 65 °C with a CO₂ flow rate of 14.48 g/min, while the highest purity for stage (55.40%) was attained at 550 bars and 65 °C with a CO₂ flow rate of 14.48 g/min. The maximum recovery of FAs, equal to 8.47 mg/g, was achieved at 550 bars and 75 °C with a CO₂ flow rate of 14.48 g/min. Moreover, the lowest biomass loading (2.45 g) and the first extraction cycle (30 min) allowed the maximum extraction of β-carotene and FAs.