There is a lack of information on pretreatments that improve the separation of biologically valuable extracts from Spirulina using supercritical carbon dioxide (scCO2). Therefore, this study was focused on testing microwave irradiation and rapid gas decompression pretreatments for the recovery of Spirulina extracts that express antioxidant, antimicrobial, and cytotoxic activity. Extractions from native and pretreated microalgae were performed using scCO2 at 40 MPa and 40 °C, without or with ethanol as a co-solvent. Results revealed that the extraction process was positively influenced to some extent by the application of each pretreatment and co-solvent addition. Specifically, extraction yield was improved up to 8.7-fold, separation of γ-linolenic acid increased from 7.3% to 35.6 %, chlorophyll A content increased from 12.1 to 272.1 mg/g extract, and tocopherol amount rose from 1.3 to 24.7 mg α-TE/g dry biomass. On the other hand, separations performed with co-solvent decreased the content of carotenoids from 100.2 to 35.6 mg/g as well as the antioxidant activity of extracts and their cytotoxicity on human hypopharyngeal squamous cell carcinoma. Moreover, the addition of ethanol to scCO2 had an adverse effect on the flavonoid content (which ranged from 45.8 to 82.4 mg RE/g) and the antimicrobial activity of extracts against 32 microbial strains. This exploratory study provides guidelines for the design, operation, and intensification of an environmentally friendly separation of added-value Spirulina extracts.