In the last decades, atomic force microscopy (AFM) has evolved towards an accurate and lasting tool to study the surface of living cells in physiological conditions. Through imaging, single-molecule force spectroscopy and single-cell force spectroscopy modes, AFM allows to decipher at multiple scales the morphology and the molecular interactions taking place at the cell surface. Applied to microbiology, these approaches have been used to elucidate biophysical properties of biomolecules and to directly link the molecular structures to their function. In this review, we describe the main methods developed for AFM-based microbial surface analysis that we illustrate with examples of molecular mechanisms unravelled with unprecedented resolution.