The development of multilayered thin film assemblies containing (bio)molecules is driven by the need to miniaturize sensors, reactors, and biochips. Viral nanoparticles (VNPs) have become popular nanobuilding blocks for material fabrication, and our research has focused on the well-characterized plant virus Cowpea mosaic virus (CPMV). In a previous study, we have reported the construction of multilayer VNP assemblies. Here we extend these studies by providing further details on the formation and properties of arrays that are made by the alternating deposition of biotinylated CPMV particles and streptavidin molecules. Array formation was followed in real time by a quartz crystal microbalance with dissipation monitoring. Our data provide indications that multiple interactions between biotin and streptavidin not only promote the assembly of a multilayered structure but also generate cross-links within each layer of CPMV particles. The degree of intralayer and interlayer cross-linking and hence the mechanical properties and order of the array can be modulated by the grafting density and spacer length of the biotin moieties on the CPMV particles.