A process for the enrichment and selective culture of mycobacteria contained in a biological sample, wherein all or part of the sample is inoculated in/on a culture medium including a nutritive component suitable for the development and growth of mycobacteria, wherein the culture medium includes, as selective agents, at least 9-chloro-9-(4′-diethylamino)phenyl-9,10-dihydro-10-phenylacridine hydrochloride (or C-390) and an agent capable of inhibiting Pseudomonas aeruginosa bacteria. The invention also relates to a culture medium suitable for implementing this process for the enrichment and selective culture of mycobacteria.