Humans are exposed to a wide range of microorganisms at birth, including bacteria, viruses, fungi, archaea, protozoa, and helminths. Preclinical and clinical evidence has linked the microbiota and its metabolites to tumor initiation, promotion, and progression. The conventional concept is that organismal microbial homeostasis is positively correlated with health, while microbial diversity or dysfunction promotes the development of diseases, including various types of cancer. Causes of dysbacteriosis include changes in genetics, environment (e.g., inflammation, drugs, diet), or pathogenic infections. However, it remains controversial whether microbial imbalance is a cause or a consequence of tumor development. Therefore, the interrogation of the role of microorganisms in tumors requires a holistic perspective. In this review, we describe the composition of cancer microbiome, progress methods of microbial detection and their role in cancer diagnosis, and provide an outlook on future research on cancer microbiome.