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Abstract Background In addition to direct slide microscopy, traditional culture method (TCM) has long been considered as a gold standard method for the diagnosis of cutaneous leishmaniasis (CL). However, TCM is relatively expensive and time-consuming compared to the newly introduced microculture method (MCM), which has shown to be sensitive and rapid diagnostic method elsewhere for different Leishmania parasite species other than Leishmania (L.) aethiopica. The objective of this study was to evaluate the diagnostic performance of MCM for the diagnosis of CL caused by L. aethiopica. Methods One hundred forty-three lesion aspirates were collected from 124 suspected CL patients prospectively based on their consecutive series. Portion of the aspirates were cultured in duplicate in TCM with modified Novy-MacNeal-Nicolle (NNN) in tissue culture flask and microcapillary tubes containing RPMI 1640 with 10% fetal bovine serum (FBS) for MCM. Smears on glass slides from the remaining portion of the aspirate were used for direct microscopy to detect the parasite after stained with Giemsa staining solution. Up on a consensus, positive result in any two of the three tests was used as a reference standard to analyze sensitivity. Results As per consensus standard criteria, 52 of the lesions were qualified to evaluate MCM versus TCM. Forty-eight lesion samples were positive by MCM, 36 by TCM, and 37 by smear microscopy. The representative DNA from parasite culture isolates revealed the causative Leishmania parasite was L. aethiopica by ITS1 polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Culturing L. aethiopica in vitro by MCM is more sensitive (92.3%) than by TCM (69.2%), P = 0.003. The median time for L. aethiopica promastigotes emergence in the culture was 3 days for MCM and 6 days for TCM, P