Recent advancements have revealed the presence of a microbiome within tumor tissues, underscoring the crucial role of the tumor microbiome in the tumor ecosystem. This review delves into the characteristics of the intratumoral microbiome, underscoring its dual role in modulating immune responses and its potential to both suppress and promote tumor growth. We examine state-of-the-art techniques for detecting and analyzing intratumoral bacteria, with a particular focus on their interactions with the immune system and the resulting implications for cancer prognosis and treatment. By elucidating the intricate crosstalk between the intratumoral microbiome and the host immune system, we aim to uncover novel therapeutic strategies that enhance the efficacy of cancer treatments. Additionally, this review addresses the existing challenges and future prospects within this burgeoning field, advocating for the integration of microbiome research into comprehensive cancer therapy frameworks.