Nanomaterials have become essential in the daily lives, finding applications in food, skincare, drugs, and vaccines. Traditionally, the surface chemistry of nanoparticles (NPs) is considered the key factor in determining their interactions with biological systems. However, recent studies have shown that the mechanical properties of nanomaterials are equally important in regulating nano-bio interactions, though they have often been overlooked. Tuning the mechanical properties of nanomaterials and designing them for biomedical applications is thus crucial. This review begins by discussing the various mechanical cues in biological processes, including how viruses and cells adjust their mechanical properties throughout their life cycles. Basic concepts and terminology related to NP mechanical properties are introduced. Next, five different groups of nanomaterials with tunable mechanical properties are explored. The review then examines the impact of NP mechanical properties on their interactions in vitro and in vivo, covering tumor-targeted drug delivery, nanovaccines, and emerging applications such as oral and intranasal drug delivery. Current challenges in the field and perspectives on future developments are also provided.