AbstractMycetoma is a chronic granulomatous inflammatory disease that is caused either by fungi (eumycetoma) or bacteria (actinomycetoma). The latter is caused by various actinomycetes of the genera Nocardia, Streptomyces and Actinomadura. They have different geographical distributions within mycetoma-endemic regions. In parts of Latin America, Nocardia species are more often encountered while in Africa, Streptomyces species dominate. For instituting a proper patient treatment plan, accurate identification of the causative organism is vital. For actinomycetoma, different laboratory-based techniques have been developed during recent decades. These include direct microscopy, cytology, histopathology and serology. More recently, different molecular techniques and matrix-assisted laser desorption ionisation-time of flight mass spectrometry have been included as diagnostic methods for actinomycetoma. In this review, an update on the laboratory techniques currently in use for the identification of actinomycetoma-causative agents to the species level is presented.