The natural capability shown by cationic vesicles in interacting with negatively charged surfaces or biomolecules has recently attracted increased interest. Important pharmacological advantages include the selective targeting of the tumour vasculature, the promotion of permeation across cell membranes, as well as the influence of cationic vesicles on drug delivery. Accordingly, cationic amphiphiles derived from amino acids may represent an alternative to traditional synthetic cationic surfactants due to their lower cytotoxicity. The importance of a synthesized lysine-based gemini surfactant (labelled C6(LL)2) was evaluated in drug delivery by designing cationic niosomes as usable pharmaceutical tools of chemotherapeutics and antibiotics, respectively like methotrexate and tetracycline. The influence of formulation factors on the vesicles’ physical-chemical properties, drug entrapment efficiency, in vitro release and ex-vivo skin permeation were investigated. A niosomal gel containing the gemini surfactant was also tested as a viable multi-component topical formulation. Results indicate that in the presence of cholesterol, C6(LL)2 was able to form stable and nanosized niosomes, loading hydrophilic or hydrophobic molecules. Furthermore, in vitro release studies and ex-vivo permeation profiles showed that C6(LL)2-based vesicles behave as sustained and controlled delivery systems in the case of parenteral administration, and as drug percutaneous permeation enhancers after topical application. Finally, cationic C6(LL)2 acts as a carrier constituent, conferring peculiar and interesting functionality to the final formulation.