A novel series of neutral and cationic dimeric surfactants were prepared involving ketalization reaction, Williamson etherification, and regioselective oxirane ring opening with primary and tertiary alkyl amines. The critical micelle concentration (CMC), effectiveness of surface tension reduction (γCMC), surface excess concentration (Γ), and area per molecule at the interface (A) were determined and values indicate that the cationic series is characterized by good surface-active and self-aggregation properties. For the first time, we reported the antimicrobial activities against representative bacteria and fungi for dimeric compounds. The antimicrobial activity was found to be dependent on the target microorganism (Gram-positive bacteria > fungi > Gram-negative bacteria), as well as both the neutral or ionic nature (cationic > neutral) and alkyl chain length (di-C12 > di-C18 > di-C8) of the compounds. The cationic di-C12 derivative was found to have equipotent activity to that of benzalkonium chloride (BAC) used as standard.