The present study deals with the formulation and characterization of bio-based oil in water nanoemulsion and its potential antibacterial activity against P.mirabilis. The nanoemulsion was formulated using eucalyptus oil, Tween 20 and ethanol by high energy method, Ultrasonication and the mean droplet size was found to be 20.17 nm as confirmed by dynamic light scattering. The nanoemulsion was highly stable, transparent and found to be effective bactericidal activity against tested pathogen. Growth inhibition was found to be 100% when treated with nanoemulsion as confirmed by dilution plate count and antibacterial susceptibility method.