Biological events such as microbial adhesion, microbial aggregation and molecular recognition play a pivotal role in the natural environment, in medicine and in biotechnological processes. Understanding the molecular bases of these phenomena requires knowledge of the structural and physical properties of microbial cell surfaces. With atomic force microscopy (AFM), it is now possible to explore the surface of single cells with nanometer lateral resolution and under physiological conditions. AFM can be used for visualizing surface ultrastructure (crystalline arrays, appendages), for following physiological changes (germination, growth) and for monitoring the effect of external agents (antibiotics, metals). These studies open the door to new applications in biotechnology and biomedicine, such as the rapid detection of microorganisms and the rationale design of drugs. AFM is actually much more than a microscope in that it also enables physical properties to be probed quantitatively: (i) surface hydrophobicity and electrical properties can be mapped using probes functionalized with defined functional groups (CH3, OH, COOH); (ii) surface softness can be measured by pressing the probe onto the cell surface; (iii) the elasticity of surface macromolecules, such as polysaccharides, can be addressed by means of force spectroscopy. These measurements have a great potential for elucidating the structure-function relationships of microbial surfaces (molecular recognition, conformational changes, surface interactions). In this contribution, I will discuss recent data obtained on fungal spores, yeasts and bacteria to highlight these unique capabilities.