Abstract: With the advancement of high-throughput sequencing technologies, the significant role of the intratumoral microbiota (ITM) in tumor initiation, progression, and treatment response has been increasingly uncovered. The ITM can promote tumor development by shaping the pro-inflammatory tumor microenvironment, inducing host DNA damage, activating oncogenic signaling pathways, and modulating immune evasion. Conversely, the ITM may also enhance antitumor immunity through mechanisms such as stimulator of interferon genes pathway activation, stimulation of T cells and natural killer cells, formation of tertiary lymphoid structures (TLS), and antigen presentation. This review summarizes the regulatory mechanisms by which the ITM influences tumor immunity and immune escape, and discusses its potential applications in cancer diagnosis, immunotherapy, radiotherapy, chemotherapy, and targeted therapy. Accumulating evidence suggests that the diversity of the ITM is closely linked to the clinical manifestations and therapeutic outcomes in cancer. Therefore, targeting the ITM may represent a promising novel strategy for cancer diagnosis and treatment.