Many obligate or facultative intracellular bacteria pose a critical problem in clinical microbiology diagnosis as a result of their fastidious growth or lack of growth in conventional culture media. Molecular diagnosis is based on the analysis and demonstration of nucleic acids (DNA and RNA). In the field of infectiology, it combines laboratory medicine with the technology of molecular genetics to identify infectious pathogens. Fluorescence in situ hybridization (FISH) is used for the detection and localization of nucleotide sequences in various samples while preserving cell integrity. For more than 30 years, FISH methods have in constant evolution with the development of rRNA-targeted probes and synthetic molecules, such as PNA, which have contributed to the development of this technique in various fields by research and diagnostic laboratories. We describe here a panel of infectious diseases due to intracellular bacteria for which FISH diagnosis has proven its effectiveness. FISH techniques were applied in cases of blood-culture-negative endocarditis, respiratory infections, gastrointestinal diseases, mycobacterial infections, highly pathogenic microorganisms and other fastidious bacteria such as spirochetes. FISH has been proven to be applicable to various samples and for diverse infectious diseases, it can be used as a complementary tool for the diagnosis of infectious diseases by intracellular and fastidious bacteria.