Abstract: As research delves deeper into the mechanisms of tumor immune responses, studies reveal the importance of microbial communities within the tumor microenvironment in tumor progression and their interactions with the host immune system. Intratumoral microbiota could influence the tumor microenvironment, thereby promoting or inhibiting tumor growth and development. Despite this importance, the specific role of intratumoral microbiota impacting cancer immunotherapeutic efficacy remains largely unexplored. A deeper understanding of the characteristics and biological functions of tumor-specific microbiota heralds a potential revolutionary innovation in cancer treatment. In this review, we introduce the discovery and sources of intratumoral microbiota, also addressing its composition, and discuss tumor tissue characteristics. Moreover, we briefly review the history of cancer immunotherapy development with a particular focus on the research progress concerning the impact of intratumoral microbiota on cancer immunotherapy. Furthermore, we explore emerging strategies that combine targeting intratumoral microbiota with immunotherapy to enhance immune efficacy, inhibit tumor progression, and improve cure rates, anticipating that this approach could represent a new direction for enhancing treatment outcomes and prospects.