Mycobacteria have unique cell envelopes, surface properties and growth dynamics, which all play a part in the ability of these important pathogens to infect, evade host immunity, disseminate and to resist antibiotic challenges. Recent atomic force microscopy (AFM) studies have brought new insights into the nanometre-scale ultrastructural and mechanical properties of mycobacteria. The molecular forces with which mycobacterial adhesins bind to host factors, like heparin and fibronectin, and the hydrophobic properties of the mycomembrane have been unravelled by AFM force spectroscopy studies. Real-time correlative AFM and fluorescence imaging have delineated a complex interplay between surface ultrastructure, tensile stresses within the cell envelope and cellular processes leading to division. The unique capabilities of AFM, which include sub-diffraction limit topographic imaging and piconewton force sensitivity, have great potential to resolve important questions that remain unanswered on the molecular interactions, surface properties and growth dynamics of this important class of pathogens.