This article will review traditional and newer microbiological techniques for the diagnosis of mycobacterial respiratory infections. It will concentrate on the diagnosis of infections due to Mycobacterium tuberculosis, the main mycobacterium causing respiratory infections of clinical and public health importance. The diagnosis of respiratory disease associated with non-tuberculous mycobacteria (NTM), particularly in children with underlying airway pathology such as cystic fibrosis (CF) or bronchiectasis, will be briefly discussed. With respect to the diagnosis of tuberculosis (TB), the review will concentrate on the diagnosis of patients with symptoms and/or signs of clinical disease, rather than the detection of exposure or asymptomatic infection. It will not specifically address the assessment of pre-test probability based on clinical or epidemiological factors, the use of radiological investigations or the investigation of extrathoracic lymph node disease or chest wall disease. The role of newer diagnostic modalities including nucleic acid detection (NAD) and gamma-interferon assays in paediatric practice will be reviewed, and suggestions made as to how they may fit into contemporary diagnostic algorithms.