Gemini cationic surfactant with tetrasiloxane-tailed has potential to improve performance and biocompatibility in a range of applications. With different rigidity spacers, structure effects on their self-assembling behavior and interface properties are obvious. Hence, it should be possible to tune system surface properties and aggregation morphology in aqueous solution by changing rigidity of spacers. A series of novel cationic tetrasiloxane Gemini surfactants MR8M, MDM, MDOM were designed and synthesized by a simple process. Their surface property, hydrolysis resistance ability, wetting ability, aggregation behavior as well as antibacterial activity in aqueous solutions were studied. These three surfactants exhibited high hydrolysis resistance in both acidic and neutral aqueous solutions. On one hand, surface tension of MR8M (γCMC = 19 mN/m) and MDOM (γCMC = 23 mN/m), significantly lower than C8-PSi-C8 (γCMC = 37 mN/m) with siloxane spacer and pure hydrocarbon equivalents. On the other hand, MR8M can wet the highly hydrophobic PTEF plate completely at low concentration (0.4 mM). In addition, different aggregations, MDM with only vesicle, MDOM with spherical micelle and vesicle micelle and MR8M spherical micelle, wormlike micelle and vesicle were observed to assemble. Moreover, the antibacterial activity of these surfactants increased as the rigidity of spacer increased and MDM exhibited best antibacterial activity.