Emerging studies have revealed the role of microbiota in regulating tumorigenesis, development, and response to antitumor treatment. However, most studies have focused on gut microbiota, and little is known about the intratumoral microbiome. To date, the latest research has indicated that the intratumoral microbiome is a key component of the tumor microenvironment (TME), and can promote a heterogeneous immune microenvironment, reprogram tumor metabolism to affect tumor invasion and metastasis. In this review, we will summarize existing studies on the intratumoral microbiome of gastrointestinal cancers and reveal their crosstalk. This will provide a better understanding of this emerging field and help to explore new therapeutic approaches for cancer patients by targeting the intratumoral microbiome.