This study concerns an investigation of oculomotor nerves via imaging semithin sections with an atomic force microscope (AFM). The study used 10 oculomotor nerves removed from individuals aged between 40 and 60 years old at time of death. We adopted semithin sections for our AFM studies to extract topographical data of the fibers of this nerve. Both light microscope and electron microscope images of the same samples were employed to augment our capabilities for orientation in the AFM analyses. Transverse sections of the oculomotor nerve, imaged via AFM, show clearly identifiable ring-like nerve fibers with the myelinated axons. The samples were simultaneously characterized by error signal mode and lateral force microscopy. These AFM-related techniques revealed no information concerning friction force and elasticity due to the presence of the embedding material (epoxy resin), but showed the myelinated nerve fibers with ring-like structures corresponding to the myelin sheaths. These are the first AFM images of physiological human oculomotor nerves, containing clear three-dimensional representations of these samples.