The progress of nanotechnology in medicine has lead to novel findings. The current strategy of developing plant based nano-scale emulsion is gaining more importance due to their uniqueness and potent applications. The current research focuses on formulation, physico-chemical characterization and antibacterial effects of Tulsi (Ocimum Sanctum) oil nano-scale emulsion system using cost-effective technique. The formulation is composed of Tulsi oil, tween 20 and water at specified concentrations. Conventional titration technique is employed in generating uniform and spherical nano-sized droplets with Z -average size of 1743 ± 025 nm. Optimization of the best formulation (H3) was done through the construction of ternary phase diagram, stability studies and physico-chemical characterization. The formulation exhibited good stability, optical clarity, low-viscosity, and polydispersity index. The nano-scale system demonstrated antibacterial activity against Staphylococcus aureus (ATCC 29213). The bio-nano-interaction caused structural modification in the pathogen and demonstrated cytoplasmic contents leakage as assessed through membrane permeability study. Hence, this bio-based formulation could be of importance for further use in various biomedical applications. Copyright © 2018 American Scientific Publishers All rights reserved