Eradication of Helicobacter pylori with traditional therapy often fails in clinical treatment. As a result, a novel efficacious therapeutic agent is strongly needed. Allitridi, a proprietary garlic derivative, has been successfully used to treat both systemic fungal and bacterial infections in China. Our previous study has shown a dose-dependent inhibitory effect of allitridi on H. pylori growth. However, the antibacterial mode of action of allitridi is still unclear. Proteomic analysis was used to study the global protein alterations induced by allitridi. A total of 21 protein spots were identified to be differentially expressed. Our results indicated that the bacteriostatic mechanism of allitridi in H. pylori can be attributed to its multitarget inhibitory effects in energy metabolism and biosynthesis including amino acid biosynthesis, protein synthesis, mRNA synthesis and fatty acid biosynthesis. Allitridi can also disturb the expression of antioxidant proteins and decrease the production of virulence factors. Western blot analysis showed that allitridi at subinhibitory concentrations can potently suppress the production of CagA and VacA. Our investigations on the antibacterial mode of action of allitridi provide an insight into the potential use of allitridi as a therapeutic agent against H. pylori infection.