<p>Microalgae are valuable sources of bioactive compounds with applications in health and nutrition. <i>Tetradesmus obliquus</i> is particularly promising due to its ability to produce lipids, carotenoids, and phytosterols, making it a candidate for integrated biorefinery approaches. Despite this potential, research on the extraction, characterization, and bioactivity of phytosterols from <i>T. obliquus</i> remains scarce. The present study optimized a sequential extraction to efficiently recover phytosterols and other high-value compounds. In the first step, supercritical CO₂ (scCO₂) at 306&#xa0;bar and 40&#xa0;°C extracted PUFAs reaching concentrations of 44.07 mg g⁻¹ extract, and carotenoids, such as lutein and β-carotene, reaching concentrations of 2.22 mg g⁻¹ extract. The second step, using scCO₂ with 5% ethanol at 400&#xa0;bar and 40&#xa0;°C recovered phytosterols, with concentration of 1594.2 ± 0.002&#xa0;µg&#xa0;g⁻¹ extract for stigmasterol, 1072.7 ± 0.00&#xa0;µg&#xa0;g⁻¹ extract for campesterol and 597.5 ± 0.00&#xa0;µg&#xa0;g⁻¹ extract for β-sitosterol. In terms of bioactivity, the carotenoid-rich extract demonstrated an antioxidant capacity of 0.191 ± 0.006&#xa0;mmol Trolox eq g⁻¹ extract by ABTS and 0.05196 ± 0.00165&#xa0;mmol Trolox eq g⁻¹ extract by ORAC. The bioactive properties of <i>T. obliquus</i>-derived phytosterols were evaluated, providing new insights into their potential applications. The phytosterol-rich extract exhibited significant α-glucosidase inhibitory activity (IC₅₀ = 111.1&#xa0;µg&#xa0;mL⁻¹), indicating potential for managing diabetes. To our knowledge, this is the first time that this bioactivity has been described for phytosterols obtained from <i>T. obliquus</i>. This study highlights the efficient and sustainable recovery of phytosterols from <i>T. obliquus</i> using an optimized biorefinery approach.</p> Graphical Abstract <p></p>