Yeast identification is becoming more important due to the emerging of yeast infections that are favored by the increase of immunocompromised patients and usage of new medical practices. Some yeast identification methods have limitations and may not cover a wide range of medically important yeast species. Thus, the development of media for yeast identification was established in this study. Three hundred and ninety-five clinical isolated yeasts from 14 species were identified using a novel developed media, combined assimilation-fermentation media (ComAF Media). The identification result was compared with a standard conventional method. In addition, esterase activity patterns on Tween 20, 40, 60, and 80 substrates were studied in some selected yeast species in order to establish a supplementary test for yeast identification. Of the 395 isolates, the percentage of correlative identification between ComAF Media and standard conventional methods was 97.97%. Only 8 isolates (2.03 %) were at variance, which were identified by ComAF Media as Candida dubliniensis. However, these 8 discrepant isolates exhibited a 100% correlation with API 20C AUX. The average cut off reading time of ComAF Media was 3 days, nevertheless, only a few species needed an extended period of incubation. The alpha-methyl-Dglucoside in ComAF Media clearly distinguished C. dubliniensis, which is incapable of utilizing this substrate, from C. albicans. In comparison with conventional methods, ComAF Media was easy to prepare and convenient for storage. Moreover, workload, time-consuming and expertise were less demanding with this media and the results of assimilation and fermentation could be detectable together. Although the pattern of esterase activity could classify various yeast species into three groups, it was not specific and could not be used alone for the identification of a species level. In conclusion, ComAF Media has the potential to be an effective test for identification of medically important yeasts and can distinguish between close related species of yeasts. The esterase activity precipitation pattern could only be a supplementary test for classifying some yeast species.