Atrophic gastritis is considered to be an antecedent to intestinal metaplasia and gastric cancer. A previous study identified that Helicobacter pylori was absent at the severe atrophic gastritis stage, and alterations in the gastric microbial composition resembled those in gastric cancer. To explore the role of the bacteria absence of H. pylori in gastric carcinogenesis, in the current study, we compared the microbiota of clinically collected H. pylori-free gastric fluids from 30 patients with non-atrophic gastritis (N) and 22 patients with severe atrophic gastritis (S). We estimated the bacterial loads in the N and S groups by colony counting in culture agar as well as by measuring the concentration of the extracted DNA. The results showed a significant increase in bacterial load in patients with atrophic gastritis in comparison to non-atrophic gastritis. Then, we analyzed the microbial communities of the gastric fluids from all 52 patients using high-throughput sequencing of 16S rRNA amplicons. The Chao 1, Shannon and Simpson diversity indexes demonstrated that the bacterial richness and diversity were not significantly different between the N and S groups. Moreover, principal component analysis illustrated that the microbiomes from the S group were more scattered. Microbiota composition analysis showed that the entire dataset was clustered into 27 phyla, 61 classes, 106 orders, 177 families, 292 genera and 121 species. At the genus level, only the abundance of Prevotella was significantly different between the N and S groups. Further analysis showed that all the higher taxonomic categories were significantly different between the N and S groups. To assess the effects of the metabolic products of Prevotella spp. on gastric cell physiology, we treated the human gastric epithelial cell line AGS with acetic acid and monitored nitric oxide (NO) production. The results showed that acetic acid at low concentrations (0.5 and 5 mu M) significantly inhibited AGS cells to secrete NO compared to phosphate buffer saline-treated control cells. These results suggest that the microbiota in non-atrophic gastritis may influence gastric epithelial cell physiology.