The invention relates to a method for the enrichment and selective culture of mycobacteria contained in a biological sample, in which all or part of the sample is sown in/on a culture means comprising a nutritive component that stimulates the development and growth of mycobacteria, characterised in that the culture medium comprises, as selective agents, at least 9-chloro-9-(4'-diethylamino)phenyl-9,10-dihydro-10-phenylacridine hydrochloride (or C-390) and an agent that can inhibit Pseudomonas aeruginosa bacteria. The invention also relates to a culture medium suitable for implementing the method for the enrichment and selective culture of mycobacteria.