New gemini pyridinium amphiphiles having alkyl chain lengths of C10, C12, C14 , and C16 and appended with hydroxyl-substituted spacers have been synthesized, characterized, and investigated for their self-assembly as well as adsorption behavior by state-of-the-art techniques such as conductometry, tensiometry, isothermal titration calorimetry (ITC), and spectrofluorometry. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) have provided excellent acumen with respect to the micellar size distribution of investigated dicationics in aqueous media. Furthermore, the interaction of these dicationics with plasmid DNA, at different charge ratios (N/P), has been studied by DLS, agarose gel electrophoresis, and ethidium bromide exclusion measurements. The cytotoxicity of these geminis has been evaluated by using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay on BV2 (microglial) and C6 glioma cell lines. It was found that the varying alkyl chain length, fashioned by ether linkage close to the headgroup, and the presence of a polar linker significantly altered the physicochemical properties of these new dicationics as compared to the properties of nonfunctionalized gemini surfactants.