The application of polyphasic taxonomic studies to identify several strains of Actinobacteria is presented. These microorganisms cause opportunistic infections relatively often (especially in immunocompromised patients), but their proper classification is difficult and they may be mistaken with other similar taxa. The article involves some clinical isolates properly identified by extended analysis, including simplified chromatographic methods (TLC, GLC). These procedures helped to establish a chemotaxonomic profile based on the well-grounded detection of chemical compounds of the bacterial cell envelope. Several components are useful taxonomic markers: the main amino acids of murein, sugars, and fatty acids and polar lipids, mainly phospholipids and glycolipids. The chemotaxonomy of Actinobacteria is a part of the contemporary classification of these microorganisms and should be applied in reference laboratories to avoid or minimize diagnostic and therapeutic error.