A microalga, Isochrysis galbana, was chosen in this study for its potent natural antioxidant composition. A broad bioactive compounds spectrum such as carotenoids, fatty acid polyunsaturated (PUFA), and antioxidant activity are described with numerous functional properties. However, most of the optimization of extraction use toxic solvents or consume a lot of it becoming an environmental concern. In this research, a Box-Behnken design with desirability function was used to prospect the bioactive composition by supercritical fluid extraction (SFE) after performing the kinetics curve to obtain the optimal extraction time minimizing operational costs in the process. The parameters studied were: pressure (20–40 MPa), temperature (40–60 °C), and co-solvent (0–8% ethanol) with a CO2 flow rate of 7.2 g/min for 120 min. The response variables evaluated in I. galbana were extraction yield, carotenoids content and recovery, total phenols, antioxidant activity (TEAC method, trolox equivalents antioxidant capacity method), and fatty acid profile and content. In general, improvement in all variables was observed using an increase in ethanol concentration used as a co-solvent (8% v/v ethanol) high pressure (40 MPa), and moderately high temperature (50 °C). The fatty acids profile was rich in polyunsaturated fatty acid (PUFA) primarily linoleic acid (C18:2) and linolenic acid (C18:3). Therefore, I. galbana extracts obtained by supercritical fluid extraction showed relevant functional ingredients for use in food and nutraceutical industries.