2D nanomaterials, particularly graphene, offer many fascinating physicochemical properties that have generated exciting visions of future biological applications. In order to capitalize on the potential of 2D nanomaterials in this field, a full understanding of their interactions with biointerfaces is crucial. The uptake pathways, toxicity, long‐term fate of 2D nanomaterials in biological systems, and their interactions with the living systems are fundamental questions that must be understood. Here, the latest progress is summarized, with a focus on pathogen, mammalian cell, and tissue interactions. The cellular uptake pathways of graphene derivatives will be discussed, along with health risks, and interactions with membranes—including bacteria and viruses—and the role of chemical structure and modifications. Other novel 2D nanomaterials with potential biomedical applications, such as transition‐metal dichalcogenides, transition‐metal oxide, and black phosphorus will be discussed at the end of this review.