This study employed wild sea buckthorn (Hippophae rhamnoides L.) fruits harvested in Qinghai Province as experimental material. Following compositional analysis of their flavonoids, the antibacterial efficacy and mechanistic pathways of flavonoids in sea buckthorn against Helicobacter pylori (H. pylori) were systematically examined through in vitro and animal model experiments. The results showed that the main flavonoids in sea buckthorn were rutin, quercetin-3-O-glucoside, quercetin, catechin, isorhamnetin, kaempferol-3-O-glucoside, kaempferol and epicatechin. Among them, quercetin, isorhamnetin and kaempferol had strong inhibitory activity against H. pylori. In vitro, sea buckthorn flavonoids inhibited the growth of H. pylori through multiple mechanisms: inducing morphological transformation from rod-shaped to spherical bacteria, promoting cell shrinkage and rupture, disrupting the cell membrane to cause leakage of intracellular macromolecules, increasing membrane permeability, and suppressing urease activity. Sea buckthorn flavonoids exert therapeutic effects on H. pylori-infected mice through multiple mechanisms, including the alleviation of gastric mucosal inflammation via the Nuclear Factor KappaB (NF-κB) signaling pathway, the down-regulation of gastrin-17 (GAS17) to suppress gastric acid production, and the up-regulation of epidermal growth factor (EGF) expression to promote gastric mucosal repair and modulate the composition of gastric microbiota. This study systematically elucidated the anti-H. pylori activity and antibacterial mechanisms of flavonoids derived from sea buckthorn fruits, providing a theoretical basis for the screening of natural antibacterial agents from this plant source.