The opportunistic pathogenExophiala dermatitidishas been frequently isolated from tropical regions of the world. However, there is no report of environmental isolation ofExophialaspp. from Malaysia. The information regarding the ecology of this microbe is important for a better understanding of the opportunism. This study aims to conduct a survey of natural distribution ofExophialaspp. in Malaysia. Forty-seven strains ofExophiala-like was isolated by using selective media. These isolates from the fields were molecularly identified based on the ITS region. The biochemical activity of these microbes was tested by conducting various tests, i.e. DNase test, proteinase activity, and urea hydrolysis. Overall, 22 strains ofE. dermatitidiswere successfully obtained and identified from burnt tree bark, oil dripped soil sample, hot spring biofilm, railway track stones, tar road contaminated with petrol hydrocarbon, drain and deep mud of Sungai Pinang besides the new discovery from pigeon droppings. A single strain ofE. heteromorphawas identified from tar road contaminated with petrol hydrocarbon. Genotypes of the isolatedE. dermatitidiswere identified by the neighbor-joining tree and grouped into Genotype A, A2 and B. The existence of new Genotype A4 was confirmed by a similar cladogram position in both neighbor-joining and maximum likelihood tree. The survival ofE. dermatitidisin the hydrocarbon contaminated environment was studied by supplying engine oil and observing the growth pattern. The results of this study suggest that the opportunisticExophialaspp. was isolated from nutrient limited and harsh conditions in the natural environment.