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Malassezia yeast belongs to the normal cutaneous flora and under certain conditions it causes seborrhoeic dermatitis (SD). There is no culture-based study about the presence and density of the Malassezia in SD patients in Serbia. Aim was to show the presence, species distribution and density of Malassezia in patients with SD on lesional skin (LS) and non-lesional skin (NLS) and healthy controls (HC) and to compare data between Serbia and other countries. The study included 70 HC and 60 patients with SD in the study group (SG). Isolation, identification and examination of density of Malassezia colony-forming units from LS and NLS were performed. Malassezia was found more frequently in the SG than in HC, 90% and 60%, respectively (P < 0.01). The most frequent isolates in SG on LS were M. slooffiae (26%), followed by M. globosa (17%) and M. sympodialis (17%). The yeast density was much higher on LS of SG than on NLS of SG or in the HC group (P < 0.05). Higher density of Malassezia was shown on LS of SG than on NLS of SG and HC. M. slooffiae is the most prevalent species in SD patients in Serbia. This study demonstrated a positive relationship between severity of SD and presence of Malassezia spp.