Microbial influence on cancer development and therapeutic response is a growing area of cancer research. Although it is known that microorganisms can colonize certain tissues and contribute to tumour initiation, the use of deep sequencing technologies and computational pipelines has led to reports of multi-kingdom microbial communities in a growing list of cancer types. This has prompted discussions on the role and scope of microbial presence in cancer, while raising the possibility of microbiome-based diagnostic, prognostic and therapeutic tools. However, additional investigation and thorough validation of cancer microbiome findings are required before this translational potential can be realized. Here we provide historical context and a conceptual framework for the so-called cancer microbiome and summarize experimental studies into tumour-associated bacteria, fungi and other microorganisms. We also discuss the current evidence for microbial colonization of tumours and their varied influence on the disease, including recent debates. Finally, we consider outstanding questions and discuss our outlook for the field.