SOME Malassezial and bacterial species may act as pathogens when exposed to certain changes in the skin microclimate causing many diseases as Pityriasis versicolor (PV).Currently available drugs have various limitations, therefore attention should be drawn towards different natural products being widely popular for antimicrobial properties. The antimicrobial potentiality of five essential oils (EOs) as well as irradiated and non-irradiated human amniotic membrane (HAM) against the growth of Malassezia furfur and Staphylococcus aureus isolated from PV patients was evaluated. Combination treatment between the most potent tested antimicrobial agents was conducted by the checkerboard technique. Results revealed that the highest antimicrobial activity was exhibited by tea tree oil (TTO) and non-irradiated HAM layered and supernatant (HAM L/S) against all the tested strains. Combination of TTO with HAMS resulted in a strong synergistic activity, significantly reduced the MICs of both agents. Moreover, the mode of action of combination treatment on cell membrane of the tested pathogens was confirmed by marked release of cellular materials that absorb at 260nm, and efflux of potassium ion. In addition, the scanning electron microscopic analysis confirmed the inhibitory effect of combination of TTO with HAMS by revealing significant morphological alterations and rupture of the cell membranes of the tested strains. These findings suggest that combination holds a broad-spectrum antimicrobial efficacy, confirming its influence on the membrane integrity and morphological characteristics of tested pathogens, which provides promising information on the potential use of TTO with HAMS for treatment of PV patients.