The objective of this research was to develop antimicrobial nanoemulsions using quillaja saponin as a natural surfactant, carvacrol as an antimicrobial agent, and medium chain triglyceride (MCT) oil as a ripening inhibitor. Oil-in-water antimicrobial nanoemulsions were fabricated by homogenizing 10 wt% oil phase (carvacrol and MCT) with 90 wt% aqueous phase (quillaja saponin in 5 mM sodium citrate buffer, pH 3.5). The physical stability and antimicrobial activity of these systems was compared to nanoemulsions formed using a synthetic non-ionic surfactant (Tween 80). Stable nanoemulsions with small mean droplet diameters (d < 150 nm) could be formed by optimizing oil phase composition and surfactant level. The antimicrobial activity of carvacrol nanoemulsions formulated with Q-Naturale® was higher than that formulated with Tween 80. Dialysis and chromatography were used to monitor the transfer of carvacrol from one hydrophobic phase to another, to mimic transport from oil droplets to bacterial cell membranes. The antimicrobial activity depended on the concentration and type of surfactant used and the ability of surfactant micelles to transfer carvacrol through the aqueous phase. Display omitted •Carvacrol nanoemulsion with small droplet size (d<150 nm) was made.•The concentration and type of surfactant had effect on antimicrobial activity.•The antimicrobial activity correlated with rate of transfer of carvacrol.