Helicobacter pylori (H. pylori) infection remains a significant global health burden, contributing to gastrointestinal pathologies such as chronic gastritis and gastric cancer. Conventional antibiotic therapies face declining eradication efficacy due to rising bacterial resistance, biofilms formation and intracellular bacterial persistence. Critically, prolonged antibiotic use inevitably disrupts the balance of gut microbiota. To address these challenges, reactive oxygen species (ROS)-driven therapy has emerged as a promising alternative that strategically exploits oxidative stress to eradicate H. pylori while preventing antibiotic resistance and preserving microbiota homeostasis. This study presents a novel ROS-generating strategy using a 9-O-octadecyl derivative of berberine (BD), a non-antibiotic agent capable of inducing ROS production, which retains the broad-spectrum antibacterial property of berberine while overcoming its inherent solubility challenges. To mitigate rapid ROS burst and host cell damage, BD was encapsulated into 18β-glycyrrhetinic acid (GA)-stabilized nanovesicles (GA/BD NVs). The nanovesicles were further functionalized with the anionic phospholipid DSPG to ensure gastric mucus penetration and arrive at the infection site. Upon oral administration, GA/BD NVs enabled sustained ROS release, inducing lethal oxidative stress in H. pylori. Additionally, the formulation effectively disintegrated biofilms, destroying both extracellular and intracellular H. pylori reservoirs. The oxidative mechanism bypasses traditional resistance pathways, effectively invalidating bacterial survival strategies. In murine H. pylori infection models, GA/BD NVs achieved a 99% bacterial clearance rates. This non-antibiotic nanotherapeutic platform not only surmounts the dual challenges of antimicrobial resistance and microbiota preservation but also opens avenues for precision oxidative therapies in infectious disease management.