Gymnanthemum amygdalinum Del is one of the medicinal plants that has been shown to have antidiabetic, antimalarial, and anticancer properties. Gymnanthemum amygdalinum leaves are expected to be able to limit the increase in free radicals caused by exposure to sunlight because they have flavonoids, which are antioxidant compounds and photoprotective compounds. The purpose of this study was to determine the optimum conditions for Gymnanthemum amygdalinum leaf extract using the ultrasonic-assisted extraction method based on the value of antioxidant activity using the ferric reducing antioxidant power (FRAP) method and the possibility that it can be used as an antibacterial against E. coli and B. subtilis using the disc diffusion test method. The ultrasonic assisted extraction method on Gymnanthemum amygdalinum leaves was carried out with 4 variations of time and amplitude conditions, among others: condition A (30 minutes, 60%); condition B (30 minutes, 65%); condition C (45 minutes, 60%); and condition D (45 minutes, 65%). The IC50 value for the ability of Gymnanthemum amygdalinum leaf extract as an antioxidant is 94.83 ± 0.04 mg/L (A); 107.44 ± 0.03 mg/L (B); 145.06 ± 0.39 mg/L (C); and 153.46 ± 0.04 mg/L (D). Ultrasonic leaf extraction using 30 minutes and 60% amplitude yielded the best results for Gymnanthemum amygdalinum (condition A). Clinical and Laboratory Standards Institute (CLSI) criteria indicate that E. coli and B. subtilis do not have particularly potent antibacterial properties. Gymnanthemum amygdalinum leaves have been shown to have antibacterial activity against E. coli and B. subtilis, with average zones of inhibition of 1 mm (A), 2 mm (B), and 1 mm (C), and 1 mm (D).