Cissus sicyoides L. is a plant used in the treatment of stroke and abscesses in popular Brazilian medicine. The aim of this work was to determine the best operational condition of the supercritical fluid extraction (SFE) of C. sicyoides with CO2 and CO2 + EtOH using the Box-Behnken design based on global yield, phenolic compounds, and flavonoids and antioxidant activity, compared to conventional extraction (Soxhlet), and to evaluate the extract's neuroprotective effect. The optimal process condition corresponded to 400 bar, 40 degrees C, and 10% EtOH. Pressure and the cosolvent percentage were the factors that influenced SFE. Extracts with increased antioxidant activity were obtained in both extractions (SFE and Soxhlet). HPTLC analysis of the extracts showed the presence of terpenes, phenolic compounds, and flavonoids. The preliminary in vivo assay showed that the extract obtained by SFE has a neuroprotective and anti-inflammatory effect in a model of focal cerebral ischemia.