Cutaneous leishmaniasis (CL) caused by the Leishmania spp. parasites, is a disease characterized by nodulo-ulcerative lesions in the skin. CL is transmitted to humans by infected sandflies during blood sucking, and is endemic in about 98 countries over the world. The demonstration of amastigotes via microscopic examination, and the growth of promastigotes in NNN (Novy-MacNeal-Nicolle) medium are gold standard methods for laboratory diagnosis. The aim of this study was to compare the biphasic NNN medium that is frequently used in routine laboratories with the biphasic nutrient medium that can be prepared easily in microbiology laboratories, for the growth of promastigotes. In the study, the aspiration fluid sample was used as clinical sample which was obtained from the skin lesion of a 47-year-old female patient admitted to İzmir Katip Celebi Ataturk Education and Research Hospital dermatology outpatient clinic and pre-diagnosed as CL. The aspirate sample taken from the lesion was evaluated with microscopy, cultivation in two different media and real-time polymerase chain reaction (Rt-PCR) methods. In microscopic examination Leishmania amastigotes were observed in Giemsa-stained smears prepared from the aspiration fluid. In Rt-PCR performed by using specific primers and probes targeting ITS1 region of Leishmania parasite, a melting-curve compatible with L.tropica was detected. For cultivation, triple inoculations of the aspirate sample into NNN (NNN + RPMI 1640 + 10% fetal calf serum) and nutrient media (nutrient agar + nutrient broth + 10% fetal calf serum) were used. The cultures were incubated at 27°C for 10 days, and the number of propagated promastigotes were counted on the third, seventh and tenth days. The growth of Leishmania promastigotes was detected in both media on the third day. The number of promastigotes grown in NNN medium on the third, seventh and tenth days were 105/ml, 106/ml and 108/ml, respectively. Those values in nutrient medium were 106/ml, 107/ml and 108/ml on the third, seventh and tenth days, respectively. Although the number of promastigotes on the third and seventh days were higher in nutrient medium than NNN medium, the number of cultivated promastigotes were equal on the tenth day. As a result, nutrient medium is considered to have an impact in the diagnosis of CL, by providing an alternative to the routine medium used and can readily be available in microbiology and parasitology laboratories with long shelf-life. It was concluded that biphasic nutrient medium could be used as a supplementary medium for diagnosis in laboratories in the absence of NNN medium or can not be provided.