The synthesis of a novel class of lysine-based cationic amphiphilic derivatives of the type N(epsilon),N(epsilon)'-bis(n-acyloxypropyl)-l-lysine methyl ester salts combining several hydroxyl functions and aliphatic chains of 12 or 14 carbon atoms is described. The compounds have one, two, three, and four alkyl chains. Aggregation in water was studied by four different methods: surface tension, conductivity, chloride ion activity, and nuclear magnetic resonance ((1)H NMR). The critical aggregation concentration value of the new surfactants depends on both the number of alkyl chains and the alkyl chain length. The formation of vesicles at low concentrations was confirmed by H(1) NMR and optical microscopy. Antimicrobial activity was determined on the basis of the minimum inhibitory concentration (MIC) values. These novel lysine-based surfactants showed moderate antimicrobial activity which may be an important advantage for many biomedical applications.