Fiber-based confocal laser scanning microscopy affords a vast field of application in medical research and clinical practice. The application of fluorescent dye allows real-time imaging of yeasts of the genus Malassezia on human skin in vivo. An Ar + -laser is used to excite the fluorescent food dye sodium fluorescein at 488 nm. Its emission is simultaneously detected in the spectral region from 500-600 nm. Topically applied fluorescein labels fungal microstructures in native habitat. Cumulative intradermal injection of the same dye enables a subsurface view of the underlying cutaneous area. In the present paper, we report the ability of the combination of in vivo confocal microscopy and sodium fluorescein application to produce real-time and high-resolution images of fungal structures on human skin. The obtained confocal images demonstrate the micro architecture of superficial Malassezia yeasts and corneocytes in horizontal plane. Taxonomic classification into different Malassezia species and observation of their colonisation patterns in native milieu brings new conclusions to fungal skin behaviour and diseases. We demonstrate advantages in clinical investigation practice over traditional laboratory routine using this non-invasive imaging tool. The described method opens new promising prospects and the possibility for further studies in fungal research.