In order to enhance essential oil's stability and water insolubility, Massoia aromatica oil nanoemulsion was formulated and tested on the planktonic growth and biofilm formation of Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans; macrophage phagocytosis and on Vero cells viability. Oil in water nanoemulsion formula was optimized by using several solvents and co-solvents composition. The stability test of the formula was conducted by using a six cycle's freeze-thaw technique. Particle size and morphology were analyzed using a particle size analyzer and transmission electron microscopy. Microbial growth, biofilm formation inhibition, and cytotoxicity assays were performed on the optimized formula by using micro dilution methods. Mice macrophage phagocytosis activities against latex and C. albicans in the presence of samples were evaluated. Massoia nanoemulsion was obtained as a transparent yellowish emulsion having 99.6-99.9% of transmittance; physically and chemically stable; showed stronger antibacterial and antibiofilm on P. aeruginosa and S. aureus, moderate to C. albicans; no significant different on phagocytic activities. The IC50 of massoia oil nanoemulsion and massoia oil towards Vero cells were 35.9μg/mL and 107.5μg/mL respectively. Massoia oil nanoemulsion can protect the stability and decreases the hydrophobicity of the oil, conserve the antimicrobial and immunomodulatory activities, but increases its cytotoxicity.