Enzymatic noncovalent synthesis enables the spatiotemporal control of multimolecular crowding in cells, thus offering a unique opportunity for modulating cellular functions. This article introduces some representative enzymes and molecular building blocks for generating peptide assemblies as multimolecular crowding in cells, highlights the relevant biomedical applications, such as anticancer therapy, molecular imaging, trafficking proteins, genetic engineering, artificial intracellular filaments, cell morphogenesis, and antibacterial, and briefly discusses the promises of ENS as a multistep molecular process in biology and medicine.