The Mycobacterium marinum group (MMG) is a class of mycobacteria that includes M. marinum, the cause of chronic systemic infections in fish. This species occasionally causes granulomatous skin lesions in humans. Other members of MMG are mycolactone-producing mycobacteria (MPM; M. ulcerans, M. shottsii and M. pseudoshottsii). The cultivation-independent approach presented in this study brings a fast and reliable alternative to classically used cultivation methods. The developed triplex erp/IS2404 qPCR assay is based on a primary species-specific erp detection, which allows enumeration of MMG in analysed samples, and secondary IS2404 detection is suitable for the differentiation of M. marinum from MPM. The detection of M. marinum in clinical specimens and in artificially contaminated tissue samples has proven its applicability for diagnostic purposes.