The aim is to evaluate the antifungal activity of Melilotus officinalis widely grown in Iraq and to identify the phytochemical responsible for this activity. The antifungal activity of the water soluble fraction was investigated using agar diffusion method. The fraction was analysed to detect the active principle using TLC, HPTLC, UV and IR spectroscopy. Melilotus officinalis of Iraq exhibited a strong antifungal activity against five diagnostic fungi in concentration of 1mg % and above. TLC of the acid hydrolysed glycoside indicates the presence of kaempferol flavonoid by comparing with standard. HPTLC, UV and IR spectroscopy confirmed the presence of this flavonoid. Melilotus officinalis widely grown in Iraq exhibited a strong antifungal activity against pathogenic fungi. Kaempferol glycoside is responsible of this potent antifungal activity as it is the major phytochemical present in the polar portion of the extract.