The tumor microenvironment is increasingly recognized as a complex ecosystem in which the intra-tumoral microbiota (comprising bacteria, fungi, viruses, etc.) plays a pivotal, underappreciated role in cancer biology. This review systematically summarizes the latest research into the origins, diversity, and functional mechanisms of intra-tumoral microbiota, emphasizing their dual roles in tumor formation, progression, and response to treatment. Using high-throughput sequencing, spatial multi-omics, and integrative bioinformatics, we identify the multifactorial origins of tumor-resident microbiota, including translocation from adjacent tissues, hematogenous dissemination, and viral genomic integration. We also examine the significant inter- and intra-tumoral microbial heterogeneity influenced by anatomical, environmental, and immunological factors, and the tissue-specific functional effects of important microbial species. We detail the mechanisms by which the intra-tumoral microbiota modulate innate and adaptive immunity through pattern recognition receptor signaling, microbial antigen presentation, and microbial metabolite production, ultimately influencing tumor microenvironment composition, immune cell dynamics, and therapeutic efficacy. Finally, we critically evaluate emerging microbiota-targeted therapeutic strategies, including engineered bacteria, antibiotics, bacteriophages, and oncolytic viruses, while outlining the technical, mechanistic, and regulatory challenges that hinder their clinical translation. Our synthesis highlights the need for rigorous multi-omics profiling, causal inference, and smart delivery systems to exploit the intra-tumoral microbiota for precision oncology. This paradigm shift offers unprecedented opportunities for personalized diagnosis and therapy, marking a new frontier in cancer research and treatment.