The culture media are employed for the isolation of various biological agents causing diseases in humans and animals. In the case of leishmaniasis, this procedure is used in an attempt to isolate the causative agent (Leishmania spp.), which is essential for confirming the diagnosis. This method is considered gold standard in the diagnosis of leishmaniasis due to its good sensitivity and specificity. It is known that the causative agents of leishmaniasis have a good growth in the culture media used for the parasitological diagnosis. In addition, the isolation of the agent allows the sample to be maintained, cryopreserved and subsequently characterized by biochemical or molecular techniques. One of the disadvantages of this method is the possibility of external contaminants (bacteria and fungi) from the lesion or the environment. Therefore, it is important that the entire sample collection and processing involve sterile material and environment, with the handling of samples in laminar flow cabinet. This methodology requires trained personnel as well as sites adapted to biosafety standards for handling microorganisms class II.