Formulating plant extracts as nanoemulsions would maximize their use in different applications. This work aimed at formulating henna ( Lawsonia inermis ) extract as nanoemulsion (HENE) and studying its antimicrobial activity on some food-borne pathogens in comparison to the bulk henna extract (HE). The active compounds in henna leaves were extracted by cold solvent extraction method using ethanol and was analyzed using high performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy (FTIR) and UV–visible spectrophotometry (UV–Vis). It was found that the extract is rich in phenolic compounds, mainly, catechin, methyl gallat, elagic acid and coumaric acid. The ultrasonic emulsification was used to prepare HENE with average droplet size of 90 nm as measured by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The antimicrobial activities of HENE on seven pathogenic bacteria ( Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa and Yersinia enterocolitica ) and seven fungi ( Trichoderma reesei, Aspergillus terreus, Penicillium digitatum, Aspergillus niger, Candida albicans, Aspergillus flavus and Saccharomyces cerevisiae ) were compared with HE and penicillin. The preservative efficacy of HENE at 0.03% and 0.05% v/v on yoghurt as a model system over a cold storage period of 15 days was studied. HENE showed higher antibacterial efficacy (P < 0.05) against B. cereus, E. coli and P. aeruginosa . The antifungal effect of HENE exceeded that of both penicillin and HE (P < 0.05), in particular, against P. digitatum, S. cerevisiae and A. terreus (P < 0.001). During storage period of yoghurt, no mold or yeast were detected. • Cold extraction of active compounds of Lawsonia Inermis. • Nanoemulsion formulation of Lawsonia Inermis extract. • Antimicrobial activity of Lawsonia Inermis extract nanoemulsion. • Lawsonia Inermis extract nanoemulsion as natural preservative.