Henna represents one of the most effective traditional remedy against multiple diseases that includes ulcer, skin disorders and infection malignancies. Identifying the active ingredient of Henna species grown in different regions of the world will definitely advance its therapeutic application. The in vitro antimicrobial screening of the Algerian henna leaves (Lawsonia inermis Linn.) was assessed against four strains of pathogenic bacteria, namely Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, and one multidrug resistant yeast namely C.albicans ATCC 10231. The henna DMSO, ethanolic and ethyl acetate leaves extracts were found to be the most active fractions against gram positive bacteria S.aureus and E.faecalis and the yeast C.albican. Such data suggests that this activity is strongly related to the presence of Lawsone in henna leaves. Inhibition of the microorganisms’ growth suggests that Algerian henna can be largely exploited in research of new antimicrobial drugs.