The long-held paradigm of solid tumors as sterile entities has been revised by the advent of advanced molecular profiling technologies, including high-throughput sequencing, spatial transcriptomics, and integrated multi-omics. These innovations have revealed tumors as dynamic, polymicrobial ecosystems, colonized by diverse communities of bacteria, fungi, viruses, and archaea. Intratumoral microbial communities display cancer-type specificity, reside in specialized microniches, and are frequently detected inside malignant and immune cells. Such close interactions enable microbes to regulate tumor development via multiple mechanisms. Microbial metabolites, such as butyrate and indole-3-aldehyde, modify epigenetic patterns, DNA damage control and cellular metabolism, affecting tumor proliferation, immune escape and metastatic capacity. Polymicrobial crosstalk among bacteria, fungi, and viruses further remodels immune cell infiltration and polarization, which modulates tumor responsiveness to chemotherapy, radiotherapy and immune checkpoint blockade. Tissue-resident and circulating microbial nucleic acid signatures act as minimally invasive biomarkers for tumor early diagnosis, prognostic assessment and patient stratification. Translational studies cover antimicrobials, engineered probiotics, bacteriophage treatments, metabolite analogs and intratumoral microbiota transplantation to improve therapeutic outcomes. Nevertheless, the field faces pressing challenges—including standardizing low-biomass sampling and sequencing workflows, robustly decontaminating data, establishing causal microbe–tumor relationships, integrating host and microbial multi-omics, and navigating regulatory and ethical frameworks. Addressing these hurdles through longitudinal cohort studies, rigorous mechanistic modeling, and cross-disciplinary collaboration will be essential to harness the tumor microbiome’s full potential and usher in a new epoch of microbiome-informed precision oncology.