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Background: The World Health Organization has identified Helicobacter pylori, a Gram‐negative bacterium responsible for several gastric disorders, as one of the pathogenic bacteria that requires newer non‐antibiotic approaches for its management. We previously demonstrated that nanostructured lipid carriers (NLC) loaded with docosahexaenoic acid (DHA‐NLC) have excellent in vitro performance against H. pylori. Materials and Methods: NLC were tested against different H. pylori strains and bacteria representative from human gut microbiota. For H. pylori, resistance development and membrane permeability assays were also performed. In vivo efficacy studies were done using an H. pylori‐infected mouse model. Microbiome analysis (16S rRNA sequencing analysis) was performed on mice feces before and after DHA‐NLC treatment. Results: NLC specifically killed different H. pylori strains by membrane disruption without inducing bacterial resistance. In vivo studies demonstrated that DHA‐NLC (2 mg/mL containing 50 μM of DHA) reduced 90%–95% of the H. pylori burden and eradicated infection in 50% of the animals when treatment was administrated ad libitum for 14 days. No significant differences were found between the administration procedure (ad libitum vs oral gavage). Also, increasing the DHA‐NLC concentration to 4 and 8 mg/mL did not translate into an improvement in antibacterial performance. Notably, gut microbiome analysis showed no alterations, highlighting the safety to the gut microbiota. Finally, no histopathological changes were reported (stomach/liver sections). Conclusions: Overall, our results emphasize DHA‐NLC as a promising approach for H. pylori infection management, since they can effectively reduce the H. pylori burden without affecting gut microbiota and, in opposition to antibiotics, without anticipating the development of resistance to this treatment. [ABSTRACT FROM AUTHOR]