Gut microbiota have been implicated in the development of cancer. Colorectal and gastric cancers, the major gastrointestinal tract cancers, are closely connected with the gut microbiome. Nevertheless, the characteristics of gut microbiota composition that correlate with gastric cancer are unclear. In this study, we investigated gut microbiota alterations during the progression of gastric cancer to identify the most relevant taxa associated with gastric cancer and evaluated the potential of the microbiome as an indicator for the diagnosis of gastric cancer. Compared with the healthy group, gut microbiota composition and diversity shifted in patients with gastric cancer. Different bacteria were used to design a random forest model, which provided an area under the curve value of 0.91. Verification samples achieved a true positive rate of 0.83 in gastric cancer. Principal component analysis showed that gastritis shares some microbiome characteristics of gastric cancer. Chemotherapy reduced the elevated bacteria levels in gastric cancer by more than half. More importantly, we found that the genera Lactobacillus and Megasphaera were associated with gastric cancer.