Novel quaternary ammonium surfactants (QUATs) derived from phenylalaninyl-proline dipeptide with chain length C12 and C14 were synthesised as potential active ingredients to be used in body cleansing formulations. The physicochemical properties and biological activities of the QUATs were determined in both single and in mixed surfactant system with either the conventional anionic surfactant sodium dodecyl sulphate (SDS) or sodium N-dodecyl prolinate. The C12 QUAT derivative showed antagonistic behaviour in both SDS and sodium N-dodecyl prolinate mixed surfactant system. Comparing the mixed system of the C12 QUAT with SDS and sodium N-dodecyl prolinate, it was found that the latter displayed better antibacterial activity together with the lower ocular irritation. The C12 QUAT-sodium N-dodecyl prolinate mixture were non cytotoxic at a concentration corresponding to its MIC value, showing that the mixture was selective towards bacterial cells rather than mammalian cell lines. Diffusion measurements showed that the sodium N-dodecyl prolinate surfactant consisted of 26 molecules per micelle in water but only 3 molecules per micelle in DMSO/water (1:1). On the other hand, C12 QUAT did not form a micelle in DMSO/Water. Membrane permeability studies of the C12 QUAT and sodium N-dodecyl prolinate showed that these surfactants are capable to penetrate into deeper skin layers to exert their antibacterial and cleansing action and hence can be used as a promising candidate as active ingredients in body wash formulations.