Bioorthogonal chemistry comprises chemical reactions that can take place inside complex biological environments, providing outstanding tools for the investigation and elucidation of biological processes. Its use in combination with nanotechnology can lead to further developments in diverse areas of biomedicine, such as molecular bioimaging, targeted delivery, in situ drug activation, study of cell–nanomaterial interactions, biosensing, etc. Here, we summarise the recent efforts to bring together the unique properties of nanoparticles and the remarkable features of bioorthogonal reactions to create a toolbox of new or improved biomedical applications. We show how, by joining forces, bioorthogonal chemistry and nanotechnology can overcome some of the key current limitations in the field of nanomedicine, providing better, faster and more sensitive nanoparticle-based bioimaging and biosensing techniques, as well as therapeutic nanoplatforms with superior efficacy.