Studies have shown that the colonisation of active microorganisms is more conducive to the development of tumour immunotherapy, but intuitive evidence regarding shaping of the tumour immune microenvironment is lacking. In this study, we used Bifidobacterium longum subsp. longum (XZ01) to intervene in a colon cancer mouse model and found that its mechanism may be related to the interaction between the spatial distribution of microorganisms and tumour immunity. Through the visualisation method we established, for the first time, we showed that harmful active bacteria such as Streptococcus and Rhodococcus specifically accumulate in the middle and upper layers of tumour tissue. These bacteria likely participate in signalling pathways that affect macrophages by directly contacting or invading the macrophages, leading to a nondifferentiated state in macrophages and the loss of some immune functions. Furthermore, the accumulation of Streptococcus and Rhodococcus fragments in the deep layer of tumour tissue likely upregulates the expression of IL-10 in tumour tissue and inhibits other immune cells, such as CD8