Gastric cancer (GC) is a malignant tumour with high morbidity and mortality worldwide, and there is an urgent need for early diagnosis and precision treatment. In recent years, the role of microbiota in the occurrence and development of GC has drawn extensive attention. Particularly, the in-depth study of GC-related microbiota by 16S rRNA sequencing technology has offered valuable tools and new perspectives for exploring the microbial characteristics of GC patients. This review systematically summarises the microbial diversity and composition of GC and non-GC samples based on 16S rRNA data, outlines the progress in identifying GC-related microbial biomarkers, explores the potential mechanisms by which diagnostic microbial biomarkers influence the development of GC, and reflects on the limitations of present studies. By integrating the current evidence, this review intends to offer a new theoretical foundation and further direction for the clinical translation of microbiota research in the diagnosis and treatment of GC.