Progress in nanomedicine relies on the development of advanced tools for imaging and manipulating biological systems on the nanoscale. Atomic force microscopy (AFM) techniques have emerged as a powerful platform for analyzing the structure, properties and functions of microbial pathogens. AFM imaging enables researchers to observe microbial cell walls in solution and at high resolution, and to monitor their remodeling upon interaction with drugs. In addition, single-molecule force spectroscopy analyzes the localization, mechanics and interactions of the individual cell wall constituents, thereby contributing to elucidate the molecular bases of cell adhesion (nanoadhesome) and mechanosensing (nanosensosome). In the future, AFM-based nanoscopy should have an important impact on nanomedicine, particularly for understanding microbe–drug and microbe–host interactions, and for developing new antimicrobial strategies.