This study aimed to determine the nano-emulsion of virgin coconut oil (n-VCO) formula that can produce the best size and zone inhibition of antimicrobial activity. The VCO was formulated with the different percentages of Tween 80 (P1: 24%, P2: 25%, P3: 26%) and sorbitol (P1: 36%, P2: 35%, P3: 34%). The particle size of the n-VCO emulsion was observed under transmission electron microscopy (TEM). The antimicrobial activity test of the n-VCO was determined by a challenge test using Salmonella Typhi (S. Typhi), Staphylococcus aureus (S. aureus), and Escherichia coli (E. coli) bacteria. The data were analysed by a one-way ANOVA (P < 0.05). The significant data were furthermore tested by Duncan's multiple ranges (SPSS v26.0). This study showed that the P3 formulation (26% Tween 80 and 34% sorbitol) produced the best n-VCO among all the treatments showing a particle size of 5-100 nm. Formulas P1 and P2 produced particle sizes of about 500-1 000 nm. The antimicrobial test showed that the P3 formula had a strong inhibitory effect on S. Typhi (7.442 ± 0.52 mm), S. aureus (8.380 ± 0.49 mm), and E. coli (6.490 ± 0.82 mm). This study concluded that the formula of the detergent strongly influences the particle size of the n-VCO. The n-VCO has enormous potential to be used as an alternative antimicrobial.