Tertiary lymphoid structures (TLSs) are essential in cancer immunotherapy, markedly improving the efficacy of immune checkpoint blockade (ICB) therapy. The presence of TLSs within the tumor microenvironment (TME) is associated with improved responses to ICB therapy, making them valuable biomarkers for predicting the efficacy of ICB therapy. Recent studies have demonstrated that diverse microorganisms-including bacteria, viruses, and eukaryotes such as fungi-impact the effectiveness of ICB treatments. Notably, studies also emphasize that specific microorganisms contribute to the development of TLSs. However, the interactions between microorganisms and TLSs are complex, and the specific mechanisms by which microorganisms impact TLS formation remain largely unknown. This review summarizes emerging advances on the formation processes of TLSs, focusing on the involved signaling pathways, and discusses how microorganisms facilitate TLS formation by affecting these pathways, chemokines, antigen presentation, and immune cells. Understanding these relationships could help in identifying microorganisms, chemokines, and signaling pathways as diagnostic or prognostic biomarkers in cancer, as well as new therapeutic approaches and potential targets for cancer treatment.