Actinobacteria, the etiologic agents of tuberculosis, actinomycosis, respiratory infections and pathological skin lesions, are also classified as hazardous biological agents at the workplace. An increased number of Actinobacteria primarily occurs at the workplaces in composting plants, agriculture, waste management facilities, libraries and museums. Robust identification of Actinobacteria requires a polyphasic diagnostic strategy including an assessment of morphological, physiological, biochemical and chemotaxonomic features as well as genotyping. Commercially available diagnostic kits often do not include bacteria isolated from the environment and therefore analyses of chemotaxonomic markers--components of peptidoglycan, fatty acids, polar lipids (phospho- and glycolipids) and isoprenoid quinones are recommended. The paper discusses a comprehensive approach to the isolation and identification of Actinobacteria, with emphasis on chemotaxonomic methods. A diagnostic procedure is exemplified by environmental strains obtained from composting plants and libraries.