Atomic Force Microscopy (AFM) has proven itself over recent years as an essential tool for the analysis of microbial systems. This article will review how AFM has been used to study microbial systems to provide unique insight into their behavior and relationship with their environment. Immobilization of live cells has enabled AFM imaging and force measurement to provide understanding of the structure and function of numerous microbial cells. At the macromolecular level AFM investigation into the properties of surface macromolecules and the energies associated with their mechanical conformation and functionality has helped unravel the complex interactions of microbial cells. At the level of the whole cell AFM has provided an integrated analysis of how the microbial cell exploits its environment through its selective, adaptable interface, the cell surface. In addition to these areas of study the AFM investigation of microbial biofilms has been vital for industrial and medical process analysis. There exists a tremendous potential for the future application of AFM to microbial systems and this has been strengthened by the trend to use AFM in combination with other characterization methods, such as confocal microscopy and Raman spectroscopy, to elucidate dynamic cellular processes.