The unique capabilities of the atomic force microscope (AFM), including super-resolution imaging, piconewton force-sensitivity, nanomanipulation and ability to work under physiological conditions, have offered exciting avenues for cellular and molecular biology research. AFM imaging has helped unravel the fine architectures of microbial cell envelopes at the nanoscale, and how these are altered by antimicrobial treatment. Nanomechanical measurements have shed new light on the elasticity, tensile strength and turgor pressure of single cells. Single-molecule and single-cell force spectroscopy experiments have revealed the forces and dynamics of receptor-ligand interactions, the nanoscale distribution of receptors on the cell surface and the elasticity and adhesiveness of bacterial pili. Importantly, recent force spectroscopy studies have demonstrated that extremely stable bonds are formed between bacterial adhesins and their cognate ligands, originating from a catch bond behaviour allowing the pathogen to reinforce adhesion under shear or tensile stress. Here, we survey how the versatility of AFM has enabled addressing crucial questions in microbiology, with emphasis on bacterial pathogens. TAKE AWAYS: AFM topographic imaging unravels the ultrastructure of bacterial envelopes. Nanomechanical mapping shows what makes cell envelopes stiff and resistant to drugs. Force spectroscopy characterises the molecular forces in pathogen adhesion. Stretching pili reveals a wealth of mechanical and adhesive responses.