Microbes with complex functions have been found to be a potential component in tumor microenvironments. Due to their low biomass and other obstacles, intratumor microbiota is poorly understood. Mucosal sites and normal adjacent tissues are important sources of intratumor microbiota, while hematogenous spread also leads to the invasion of microbes. Intratumor microbiota affects the progression of tumors through several mechanisms, such as DNA damage, activation of oncogenic pathways, induction of immunosuppression, and metabolization of drugs. Notably, in different types of tumors, the composition and abundance of intratumor microbiota are highly heterogeneous and may play different roles in the progression of tumors. Because of the concern in this field, several techniques such as omics and immunological methods have been used to study intratumor microbiota. Here, recent progress in this field is reviewed, including the potential sources of intratumor microbiota, their functions and related mechanisms, and their heterogeneity. Techniques that can be used to study intratumor microbiota are also discussed. Moreover, research is summarized into the development of strategies that can be used in antitumor treatment and prospects for possible future research in this field.