The successful identification of fungi by phenotypic methods or molecular technique depends mainly on the using an accurate method for their purification. There are many methods for purifying fungi. The most efficient is the single spore technique because it is easy to carry out and effective to prevent the contamination by bacteria, yeast and mites. This technique depends on the using light microscope to transfer one spore into a new culture medium. The present work describes a simple and efficient procedure for single spore isolation to purify of fungi recovered from the clinical wastes.