Microorganisms have been identified in tumor specimens for over a century. It is only in recent years that tumor-associated microbiota has become a rapidly expanding field. Assessment techniques encompass methods at the frontiers of molecular biology, microbiology, and histology, requiring a transdisciplinary process to carefully decipher this new component of the tumor microenvironment. Due to the low biomass, the study of tumor-associated microbiota poses technical, analytical, biological, and clinical challenges and must be approached as a whole. To date, several studies have begun to shed light on the composition, functions, and clinical relevance of the tumor-associated microbiota. This new piece of the tumor microenvironment puzzle could potentially change the way we think about and treat patients with cancer.