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In this article, microalgae <i>Nannochloropsis</i> sp. was used for fatty acid (FA) extraction, using a supercritical fluid-carbon dioxide (SF-CO₂) extraction method. This study investigated the influence of different pre-treatment conditions by varying the grinding speed (200&#8722;600 rpm), pre-treatment time (2.5&#8722;10 min), and mixing ratio of diatomaceous earth (DE) and <i>Nannochloropsis</i> sp. biomass (0.5&#8722;2.0 DE/biomass) on FAs extraction. In addition, the effect of different operating conditions, such as pressure (100&#8722;550 bar), temperature (50&#8722;75 &#176;C), and CO₂ flow rate (7.24 and 14.48 g/min) on eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) recovery, was analyzed. Experimental data evidenced that, keeping constant the extraction conditions, the pre-treatment step enhanced the FAs extraction yield up to 3.4 fold, thereby the maximum extracted amount of FAs (61.19 mg/g) was attained with the pre-treatment with a ratio of DE/biomass of 1 at 600 rpm for 5 min. Moreover, by increasing both SF-CO₂ pressure and temperature, the selectivity towards EPA was enhanced, while intermediate pressure and lower pressure promoted DHA recovery. The highest amount of extracted EPA, i.e., 5.69 mg/g, corresponding to 15.59%, was obtained at 75 &#176;C and 550 bar with a CO₂ flow rate of 14.48 g/min, while the maximum amount of extracted DHA, i.e., ~0.12 mg/g, equal to 79.63%, was registered at 50 &#176;C and 400 bar with a CO₂ flow rate of 14.48 g/min. Moreover, the increased CO₂ flow rate from 7.24 to 14.48 g/min enhanced both EPA and DHA recovery.