Intra-tumoral microbes have been revealed to exist in many cancer types, attracting widespread attention. The significance of intra-tumoral microbes is becoming increasingly apparent in various aspects of human cancers, encompassing cancer initiation, progression, metastasis, diagnostic approaches, prognostic evaluations, and therapeutic interventions. Despite the considerable focus dedicated to this topic by numerous scholars, a comprehensive analysis of intra-tumoral microbiota is still lacking in human cancers. Especially, identifying specific microbial hallmarks in the occurrence and development of cancer and different cancers remains the central task for investigators. This review focuses on the identification and analysis of distinct attributes and noteworthy characteristics exhibited by intra-tumoral microbiota across various types of cancer. The potential mechanisms of intra-tumoral microbiota action, as well as the significance of the microbiome in the diagnosis and prognosis of cancer, are systematically summarized. The capacity of intra-tumoral microbes to regulate cancer treatment with a focus on the relevant microbial species, and the possibility of targeting the microbiota to improve treatment effectiveness while preventing toxicity, are specifically highlighted. Lastly, the challenges, limitations, and prospects of intra-tumoral microbes in further study and clinical application, including prognostic, diagnostic, and therapeutic applications, are discussed in cancers. This review provides a systematic summary of the specific characteristics, molecular mechanisms, therapeutic effects, and diagnostic and prognostic values of intra-tumoral microbiota in different cancers, which will help improve the diagnosis, treatment, and prognosis of tumor patients and offer new ideas for achieving precise treatment of cancer with intra-tumoral microbiota.