Hancornia speciosa has bioactive molecules such as phenolic compounds which exhibit antioxidant and antimicrobial properties. Nevertheless, the separation of these substances from a medicinal plant can be different according to the extraction process and solvent used. Thus, the present study aimed was to separate phenolic compounds from Hancornia speciosa leaves in three steps by using a sequential pressurized liquid extraction and evaluated their antioxidant and antibacterial activity. Results showed that the temperature increase from 25 to 60 degrees C provides a significative enhancement in the extraction yield. The raw ethanol extract (39.0 +/- 0.2%) showed the highest overall extraction yield. However, fractionated ethanol extract (FEE) at 60 degrees C exhibited the highest amount of total phenolic compounds (347.0 +/- 4.12 mg/g extract). Fractionated ethyl acetate extract (FAE) at 60 degrees C presented improved quantity of total flavonoids (230.4 +/- 3.5 mg/g extract) and rutin content (16.6 +/- 0.3 mg/mL extract). The antioxidant activity results for IC50 of DPPH indicated that FEE (3.8 +/- 0.1 mu g/mL) was the best free radical scavenger. FAE and FEE were able to inhibit the growth of multidrug-resistant E. coli.