We have investigated the effect of salt additives (NaCl, Na2SO4, Na3PO4, NaTos, and NaAn) on the aggregation behavior of a cleavable biodegradable ester-bonded dicationic gemini surfactant, ethane-1,2-diyl bis(N,N-dimethyl-N-hexadecylammoniumacetoxy) dichloride (16-E2-16). A multitechnique approach employing tensiometry, fluorescence, proton magnetic resonance (H-1 NMR), transmission electron microscopy (TEM), absorption spectrophotometry (UV), and Fourier transform infrared spectroscopy (FTIR) was utilized to probe physicochemical fluctuations. Appreciable changes were observed in various physicochemical parameters, viz., critical micelle concentration (CMC), surface excess concentration (Gamma(max)), minimum area per headgroup (Amin), free energy of micellization (Delta G(mic)), free energy of adsorption (Delta G(ads)), and aggregation number (N-agg). Counter ions were found to affect through electrostatic and hydrophobic influence obeying the overall trend as NaAn > NaTos > Na3PO4 > Na2SO4 > NaCl. H-1 NMR, TEM, UV, and FTIR results reveal microstructure evolution and phase transitions. These results thus provide deeper insights in understanding of self-aggregation and microstructure evolution of biocompatible (green) aqueous systems of the gemini surfactant and their implications in the biomedical and pharmaceutical world, which could be helpful to improve their bioavailability and other biochemical aspects like drug delivery and gene transfection.