Atomic force microscopy (AFM) has recently opened a variety of novel possibilities for imaging and manipulating microbial surfaces in their native environment. While AFM imaging offers a means to visualize surface structures at high resolution and in physiological conditions, AFM force spectroscopy enables researchers to probe a variety of properties, including the unfolding pathways of single-membrane proteins, the elasticity of cell walls and surface macromolecules, and the molecular forces responsible for cell-cell and cell-solid interactions. These nanoscale analyses enable us to answer a number of questions that were difficult to address previously, such as: how does the surface architecture of microbes change as they grow or interact with antibiotics; what is the force required to unfold and extract a single membrane protein; and what are the molecular forces driving the interaction between a pathogen and a host or biomaterial surface? This review will expand on these issues.