<p>Microorganisms play critical roles in tumor development, diagnosis, and treatment. In recent years, the newfound understanding of microorganisms in cancer tissues has sparked a renewed enthusiasm and research interest. The intratumoral microbes and metabolites have been discovered to interact with tumor cells to exert tumor-promoting or tumor-suppressing effects. Intratumoral microorganisms and their metabolites can alter tumor metabolism, activate key signaling pathways, regulate anti-tumor immunity, and affect tumor metastasis or recurrence. Additionally, intratumoral microbiota has emerged as promising predictive biomarkers for both diagnosis and prognosis of cancer patients. Intratumoral microorganisms and metabolites also have a substantial impact on tumor treatment by influencing therapeutic efficacies of immunotherapy, chemotherapy and radiotherapy. Intratumoral microbes can also serve as carriers to transport anti-tumor drugs, directly modulating intratumoral microecology and intervening in the carcinogenic pathway. However, the current research on the understanding of intratumoral microbiome and their metabolites is still limited and warrants more in-depth exploration. In this review, we extensively explore the structure and role of the intratumoral microbiota and metabolites in different cancer types, summarize the potential role in tumor diagnosis and prognosis, and provide recent advances of clinical implications for cancer treatment. (Source: Created with https://www.biorender.com/)</p>