The recent developments in virus research and the application of functional viral particles in nanotechnology and medicine rely on sophisticated imaging and manipulation techniques at nanometre resolution in liquid, air and vacuum. Atomic force microscopy (AFM) is a tool that combines these requirements and imaging of viruses dates back to the early days of AFM. AFM has a comparable resolution to electron microscopy, but has the advantage that it can be used to image in fluid. This implies that dynamic processes in physiologically relevant environments can be studied and one of the first examples of AFM imaging of viruses was the real-time observation of viral infection of living cells. Improvement of the imaging techniques led the way to image fragile viruses in non-destructive ways, to visualise viral capsomeres in liquid and to image genome uncoating. In addition to imaging, AFM is also used as a tool to manipulate viral nanoparticles. Translation, rotation and nano-dissection of viruses are possible and by performing nanoindentation experiments the viral material properties can be examined. Next to providing the Young's modulus and breaking force of viral shells, such experiments have also elucidated the impact of the genome on the overall viral mechanical properties and the effect of capsid structural modifications. The combination of modelling and AFM experiments finally, yields a deeper insight into their structure, function and behaviour. Here we review the early AFM experiments on viruses, the achievements made since then and the recent advances in imaging and manipulation. © 2010 The Royal Society of Chemistry.