Recently, a variety of patterning techniques have reached feature sizes of 100 nm or less, a size range very relevant to biology. Proteins, vesicles, and macromolecular assemblies can now be handled and specifically placed onto predefined artificial patterns, triggering defined functions in cells and revealing the details of cell–surface interactions. Simultaneously, novel surface chemistries have been developed that are able to induce specific bioresponses (e.g., mimicking the features of the extracellular matrix) and at the same time suppress the nonspecific effects of complex biological solutions. This article reviews the basic principles and properties of multifunctional physisorbed monolayers that can be used in combination with nanopatterning techniques to create biologically relevant surface features. Furthermore, selected examples of nanopatterns created by novel combinations of different top-down and bottom-up approaches are presented, including systems with specific bioligands, proteins, vesicles, and cells.