The intratumoral microbiome (TM) refers to the microorganisms within tumor tissue, including bacteria, fungi, viruses, etc., as distinct from the gut microbiome and circulating microbiome. TM is closely related to the occurrence, development, metastasis and treatment response of tumors. This paper focuses on the research status of TM. Duct origin, adjacent normal tissue, circulatory system, and concomitant tumor co-metastasis are the main sources of TM. A comprehensive summary of the advanced technology of TM analysis. In addition, TM is involved in tumor progression through multiple mechanisms, Including DNA damage, activation of carcinogenic signaling pathways (phosphoinositol 3-kinase [PI3K], signal transduction and transcriptional activator [STAT], WNT/β-catenin and extracellular regulatory protein kinase [ERK]), cytokine influence and induction of inflammatory responses, And interaction with the tumor microenvironment (anti-tumor immunity, pro-tumor immunity, and microbially derived metabolites). In addition, promising directions for TM in oncology therapy include immunotherapy, chemotherapy, radiotherapy, probiotic/prebiotic/bibiogenesis applications, fecal microbiome transplantation, engineered microbiota, phage therapy, and oncolytic virus therapy. The challenges inherent in clinical applications are also summarized. This review provides a comprehensive overview of the analysis of TM, in particular the mechanisms associated with TM and TM-based cancer therapies.