The current treatments of acne vulgaris and acne-like disorders such as gram-negative folliculitis possess lots of unwanted side effects. Thus, alternative approach of utilizing natural plant extracts, specifically Piper betle L., have gained much attention. To this end, this work developed, characterized, and ex vivo evaluated novel antibiotics hydrogels containing P. betle L. leaf extract for acne treatment. Firstly, the design of experiments (DoE) D-optimal method was successfully developed, optimized, and validated, to investigate the relationship between P. betle L. extraction conditions and the extract properties. Secondly, the best extract was encapsulated in the hydrogel formulations composed of carbopol 940, propylene glycol, and cocamidopropyl betaine. Finally, the hydrogel was ex vivo determined its antibacterial activity on bacteria isolated from 15 patient acne samples. The optimal extraction condition being an extraction solvent/plant weight ratio of 4.034:1, an extraction time of 2.147 h, and a water extract volume of 91.4 mL. This condition yielded an extract total phenolic content of 3.337±0.034 g GAE/g, and minimum inhibitory concentrations of 32 µg/mL and 128 µg/mL on Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922, respectively. The hydrogel possessed suitable properties for a topical medication, including a viscosity of 6800 cps, a pH of 7.0, and a good foaming ability, at both 10°C, 25°C, and 40°C. The hydrogel showed higher antibacterial activity than the positive controls in both gram-positive and gram-negative bacteria. Conclusively, the hydrogel could become a potential pharmaceutical product for acne treatment.