An understanding of the complex nomenclature and 3D spatial relations between the cortical and white matter components of the retrocommissural portion of the hippocampal formation is essential for a successful outcome when performing surgery in the mediobasal temporal region. The goal of this study was to clarify the nomenclature related to the retrocommissural portion of the hippocampal formation and to provide a detailed description of its topography and inner structure from a relevant surgical perspective. This description can serve as an anatomical reference for approaching lesions in the mediobasal temporal region. Fiber microdissection was performed from the superolateral and inferior aspects on 20 previously frozen, formalin-fixed human brains. Three formalin-fixed plastinated brain specimens were then sectioned in the coronal, axial, and sagittal planes, and the relevant sections were studied. Based on its relationship with the corpus callosum, the hippocampal formation is subdivided into precommissural, supracommissural, and retrocommissural sections. The retrocommissural portion of the hippocampal formation, a structure in the mediobasal temporal region, is a component of both the floor of the temporal horn and the medial surface of the cerebral hemisphere. It includes the hippocampus (Ammon's horn and the dentate gyrus), subiculum, and related white matter fibers. Step-by-step microdissection revealed the complex ventricular and cisternal relationships of the retrocommissural portion of the hippocampal formation in the mediobasal temporal region. This further allowed clear distinction of each component of this formation in order to understand their complex reciprocal relationships that comprise a unique internal architecture and external morphology. The fiber microdissection technique provides a valuable perspective for understanding the complex structure and relations of the components of the retrocommissural portion of the hippocampal formation. This study clarifies and supplements the information presently available in the literature by offering an anatomical reference essential for differentiating the topographic diagnosis and safer surgical planning for any lesion within this formation or the mediobasal temporal region.