The bioactive compounds were extracted from Annona squamosa leaves using ultrasound-assisted extraction (UAE) and the optimal extraction conditions were determined. It was found that the extraction of the sample with 50% (aq) of ethanol at 50 °C for 60 minutes provided the highest crude extract yield of 30.50% on a dry basis. Further, this condition gave a total phenolic content of 307.67 μg GAE/g DW and total flavonoid content of 16,893.92 μg CE/g DW. The crude extract obtained from this condition gave the best results for antioxidant activities with the IC50 values of 947.99 and 620.89 μg/mL by DPPH and ABTS assays, respectively. The chemical composition of the crude extract was analysed by LC-ESI-MS, Rutin and Norisocorydine were detected. Finally, the antibacterial properties of the crude extract were tested against three human pathogenic bacterial strains. Results revealed that the crude extract exhibited antibacterial activities against all tested strains.