Palmitoyl tripeptide (M330) showed higher antimicrobial activities than methyl paraben or phenoxy ethanol through minimum inhibitory concentration (MIC) test. However, when the M330 was added into cosmetic formulation, white precipitates formed due to the electrostatic interaction between M330 and carbopol (carboxy vinyl polymer) as a thickener in cosmetics, and the viscosity of cosmetics decreased sharply. Also, the antimicrobial activities of M330 in cosmetics became lower than those of methyl paraben or phenoxy ethanol. Thus, the encapsulation of M330 in ethosome vesicle was attempted in order to recover the declined antimicrobial activities of M330 in cosmetics and prevent the precipitates from forming. When ethosome-encapsulated M330 was added into cosmetics, the precipitates did not form, and the decrease in the viscosity of cosmetics was not large compared to the addition of unencapsulated M330. Challenge tests showed that antimicrobial activities against gram negative bacteria were improved by the encapsulation of M330, but the encapsulation was not effective against gram positive bacteria and fungus. A combination of M330 with EDTA showed synergistic inhibitory potential against C. albicans. After coencapsulation of M330 and EDTA in ethosome, antimicrobial activities proved to be higher than those of unencapsulated M330 and EDTA.