Using molecular methods, infectious organisms of viral, bacterial and fungal origin, as well as protozoa and helminths, can be detected. Molecular methods detect specific segments in the nucleic acid sequences of infectious agents and therefore do not require the maintenance of viability of the microorganisms of interest. Therefore, these methods can also be used for direct detection of infectious agents from fixed tissue, the most commonly available material in pathology. This short review article is based on more than 20 years of molecular microbiology within pathology and our aim is to present the possibilities of molecular detection of infectious organisms for pathological diagnosis.