Atomic force microscopy (AFM) provides a unique capability to image high-resolution architecture and structural dynamics of pathogens (e.g., viruses, bacteria, and bacterial spores) at near-molecular resolution in native conditions. Further development of atomic force microscopy to enable the correlation of pathogen protein surface structures with specific gene products is essential to understand the mechanisms of the pathogen life cycle. We applied an AFM-based immunolabeling technique for the proteomic mapping of macromolecular structures through the visualization of the binding of antibodies, conjugated with nanogold particles, to specific epitopes on Bacillus spore surfaces. This information is generated while simultaneously acquiring the Surface morphology of the pathogen. The immunospecificity of this labeling method was established through the utilization of specific polyclonal and monoclonal antibodies that target spore coat and exosporium epitopes of Bacillus atrophaeus and Bacillus anthracis spores.