Nanomedicine is a discipline that applies nanoscience and nanotechnology principles to the prevention, diagnosis, and treatment of human diseases. Self-assembly of molecular components is becoming a common strategy in the design and syntheses of nanomaterials for biomedical applications. In both natural and synthetic self-assembled nanostructures, molecular cooperativity is emerging as an important hallmark. In many cases, interplay of many types of noncovalent interactions leads to dynamic nanosystems with emergent properties where the whole is bigger than the sum of the parts. In this review, we provide a comprehensive analysis of the cooperativity principles in multiple self-assembled nanostructures. We discuss the molecular origin and quantitative modeling of cooperative behaviors. In selected systems, we describe the examples on how to leverage molecular cooperativity to design nanomedicine with improved diagnostic precision and therapeutic efficacy in medicine.