Hyssopus officinalis (Lamiaceae) thrives in the mountainous regions of Algeria, where its aromatic properties and medicinal uses have been embraced by local. Traditionally used in herbal medicine, this aromatic plant has garnered attention for its potential benefits in treating infectious diseases and gastrointestinal issues, including ulcers. This study seeks to evaluate, in vitro, the antibacterial capacity of leaf η-butanol extract of Hyssopus officinalis (η-BLHE) against four pathogenic bacteria, investigate, in vivo, its protective effects on gastric mucosa, and identify its bioactive compounds. The agar well diffusion method was employed to assess the antibacterial effects. η-BLHE was orally administered to two experimental groups of rats, both before and after the induction of gastric ulcers using absolute ethanol. The extent of gastric mucosal lesions was assessed through macroscopic and histopathological examinations. Phytochemical compounds were recognized by UPLC-ESI-MS/MS.