The advent of atomic force microscopy (AFM) has recently opened a wide range of novel possibilities for probing microbial cell surfaces on the nanoscale.@footnote 1@ Using AFM imaging in aqueous solution, microscopists can visualize cell surface nanostructures (surface layers, appendages), follow physiological changes (germination, growth) and monitor the effect of external agents (antibiotics, metals) in real-time.@footnote 2@ Further, using force spectroscopy, researchers can learn about local biomolecular interactions and physical properties. Spatially resolved force mapping offers a means to determine physical/chemical heterogeneities at the subcellular level, thereby providing complementary information to classical characterization methods. Force measurements allow the cell surface elasticity to be determined.@footnote 3@ Functionalizing the AFM tip with chemical groups or biomolecules enables quantitative measurements of surface charge,@footnote 4@ surface hydrophobicity@footnote 5@ and receptor-ligand interactions. Finally, single-molecule force spectroscopy can be applied to cell surface molecules to gain insight into their mechanical properties.@footnote 6@ Clearly, these new AFM-based experiments contribute to improve our understanding of the structure-function relationships of microbial cell surfaces and will have considerable impact on biotechnology and medicine. @FootnoteText@@footnote 1@Y.F. Dufrene, J. Bacteriol., 184, 2002, 5205-5213. @footnote 2@Y.F. Dufrene, C.J.P. Boonaert, P.A. Gerin, M. Asther, P.G. Rouxhet, J. Bacteriol., 181, 1999, 5350-5354. @footnote 3@H.C. van der Mei, H.J. Busscher, R. Bos, J. de Vries, C.J.P. Boonaert, Y.F. Dufrene, Biophys. J., 78, 2000, 2668-2674. @footnote 4@F. Ahimou, F.A. Denis, A. Touhami, Y.F. Dufrene, Langmuir, 18, 2002, 9937-9941. @footnote 5@Y.F. Dufrene, Biophys. J., 78, 2000, 3286-3291. @footnote 6@B.C. van der Aa, R.M. Michel, M. Asther, M.T. Zamora, P.G. Rouxhet, Y.F. Dufrene, Langmuir, 17, 2001, 3116-3119.