The human specific bacterial pathogen Helicobacter pylori (H. pylori) is associated with severe gastric diseases, including gastric cancer. Recently, the increasing resistance makes the usage of antibiotics less effectively. Therefore, development of a new antimicrobial agent is required to control H. pylori infection. In the current study, the inhibitory effect of β-caryophyllene on H. pylori growth, as well as the antibacterial therapeutic effect, has been demonstrated. β-caryophyllene inhibited H. pylori growth via the downregulation of dnaE, dnaN, holB, and gyrA and also downregulated virulence factors such as CagA, VacA, and SecA proteins. β-caryophyllene inhibited expression of several T4SS components, so that CagA translocation into H. pylori-infected AGS gastric cancer cells was decreased by β-caryophyllene treatment. β-caryophyllene also inhibited VacA entry through the downregulation of T5_aSS. After β-caryophyllene administration on Mongolian gerbils, the immunohistochemistry (IHC) and Hematoxylin&Eosin stains showed therapeutic effects in the treated groups. Hematological data, which was consistent with histological data, support the therapeutic effect of β-caryophyllene administration. Such a positive effect of β-caryophyllene on H. pylori infection potently substantiates the natural compound as being capable of being used as a new antimicrobial agent or functional health food to help patients who are suffering from gastroduodenal diseases due to H. pylori infection.