Notwithstanding the advantages of providing definitive diagnoses, the identification of fungi based on histopathological determination can be difficult and may lead to a pit of diagnoses. Therefore, the establishment of an auxiliary diagnostic method for use in routine pathological laboratories is desirable and should improve the above situation. Our previous studies have shown the superiority of in situ hybridization (ISH) for the detection of pathogenic fungi in histological specimens. This review focuses on the usefulness of ISH in the detection and identification of pathogenic fungi from formalin-fixed and paraffin-embedded (FFPE) tissue sections, and provides an overview of ISH for the diagnosis of fungal infection and retrospective autopsy analysis using molecular procedures. Based on the above, peptide nucleic acid (PNA) probes were shown to be superior in terms of the detection of target fungi and useful since histopathological diagnosis has the potential danger of being incorrect in the identification of fungi. In conclusion, we wish to emphasize that histopathological diagnosis in combination with molecular methods such as ISH and polymerase chain reaction (PCR) of FFPE samples should enhance the accuracy of identification in relation to fungi.