Abstract: This study aims to evaluate the modified Novy-MacNeal-Nicolle (NNN) medium by adding mycosel agar to inhibit the growth of fungi and comparing it to traditional culture for the isolation of Leishmania parasites from cutaneous lesions of patients presenting to a specialized leishmaniasis clinics in Iran. A total 128 (70 female and 58 male) consecutive patients aged 3 months to 75 years (mean age of 11.25 years), presenting to Lesion biopsies were cultured in duplicate and parallel in traditional culture tubes containing NNN medium and modified NNN medium. The contamination rate in two groups of media was assessed. After sufficient incubation we have investigated that the media tubes for the presence of fungi. Through 90 samples, in 46 NNN media and 58 mycoNNN promastigotes were growth and culture positive. The difference between NNN medium and Mycosel NNN was quite significant (p<0.05). The overall sensitivity of Modified NNN medium were 75%, while traditional culture had sensitivity of 64.8%. We have demonstrated that modified NNN medium is a more sensitive and time-efficient means of isolating Leishmania parasites from cutaneous lesions than traditional culture. Our study showed that contamination of NNN medium as base culture medium for isolation of Leishmania agents could be diminished significantly by addition of mycosel agar to NNN medium.