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The present study explored the differences in gastric microbiome between three distinct populations of Southeast Asia. These include the isolated Orang Asli population and modern Malaysians, as well as patients from Myanmar, the least developed country in the region. All 79 subjects recruited in this study had Helicobacter pylori infection. Based on alpha diversity analysis, Orang Asli had the richest and most diverse gastric microbiome, followed by Myanmar and modern Malaysian groups. Beta diversity analysis revealed significant separation of samples between different populations. These observations are likely to be associated with the level of modernization of each population. Our data further suggested increased bacterial species richness and diversity of the gastric microbiome in individuals who were less modernized, particularly in the Orang Asli group, could suppress the growth of H. pylori. In addition, there were significant variations in the gastric microbiome between modern Malaysians with different types of gastric diseases. Notably, Cutibacterium acnes was present at significantly greater abundance level in patients with non-ulcerative dyspepsia than those with peptic-ulcer diagnosis. This suggests that C. acnes may also play a role in gastritis besides H. pylori, which merits further investigation.