Anise oil was prepared in its nanoemulsion form to facilitate the penetration of microbial walls, causing microbe mortality. The penetration occurred easily owing to the reduction in its size (nm). Nanoemulsions with different concentrations of anise oil were prepared using lecithin as an emulsifying agent with the aid of an ultra-sonification process. Their morphological and chemical properties were then characterized. The promising constituents were l-Menthone (11.22%), Gurjunene (6.78%), Geranyl acetate (4.03%), Elemene (3.93%), Geranyl tiglate (3.53%), geraniol (3.48%), linalool (0.17%) as well as camphene (0.12%). Different concentrations of prepared anise oil in micro and nanoemulsions were tested as antimicrobial agents against Gram-positive bacteria (Staphylococcus aureus), Gram-negative bacteria (Escherichia coli), yeast (Candida albicans) and fungi (Asperigillus niger). The findings illustrated that the anise oil-based nanoemulsion exhibited better results. Different biochemical and biological evaluations of anise oil nanoemulsions were conducted, including determining killing times, antioxidant activities (using three different methods), and total phenolics. A trial to estimate the mode of action of anise oil-based nanoemulsion as an antimicrobial agent against S. aureus and C. albicans was performed via studying the release of reducing sugars and protein and conducting scanning electron microscopy.