Electron microscopy is one of the tools required to characterize cellular structures. However, the procedure is complicated and expensive due to the sample preparation for observation. Atomic force microscopy (AFM) is a very useful characterization technique due to its high resolution in three dimensions and because of the absence of any requirement for vacuum and sample conductivity. AFM can image a wide variety of samples with different topographies and different types of materials. AFM provides high-resolution 3D topography information from the angstrom level to the micron scale. Unlike traditional microscopy, AFM uses a probe to generate an image of the surface topography of a sample. In this protocol, the use of this type of microscopy is suggested for the morphological and cell damage characterization of bacteria fixed on a support. Strains of Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), and Pseudomonas hunanensis (isolated from garlic bulb samples) were used. In this work, bacterial cells were grown in specific culture media. To observe cell damage, Staphylococcus aureus and Escherichia coli were incubated with different concentrations of nanoparticles (NPs). A drop of bacterial suspension was fixed on a glass support, and images were taken with AFM at different scales. The images obtained showed the morphological characteristics of the bacteria. Further, employing AFM, it was possible to observe the damage to the cellular structure caused by the effect of NPs. Based on the images obtained, contact AFM can be used to characterize the morphology of bacterial cells fixed on a support. AFM is also a suitable tool for the investigation of the effects of NPs on bacteria. Compared to electron microscopy, AFM is an inexpensive and easy-to-use technique.