Supramolecular chemistry has emerged as an important technique for the formation of biomaterials, including nano- and microparticles and hydrogels. One specific class of supramolecular chemistry is the direct association of guest–host pairs, which involves host macrocycles such as cyclodextrins and cucurbit­n­urils and a wide range of guest molecules, where association is typically driven by molecule size and hydrophobicity. These systems are of particular interest in the biomedical field due to their dynamic nature, chemical diversity, relative ease of synthesis, and ability to interact with biological or synthetic molecules. In this review, we discuss aspects of polymeric material assembly mediated by guest–host interactions, including the fundamentals of assembly into functional biomedical materials. Additionally, applications of biomaterials that utilize guest–host interactions are discussed with a focus on injectable material formulations, the sequestration and delivery of encapsulated cargo (i.e., drugs, biomolecules), and the investigation of cell–material interactions (i.e., adhesion, differentiation, and delivery). While methodologies for guest–host mediated assembly and biological interaction have rapidly evolved in recent years, they remain far from realizing their full potential in the biomaterials field.