A library of imidazolium‐based gemini cationic bola amphiphiles was synthesized using a regioselective ring‐opening reaction of glycidyl phenyl ether with imidazole under solvent‐free conditions. The corresponding hexafluorophosphate (PF6−) and tetrafluoroborate (BF4−) counterion‐containing amphiphiles were also synthesized and characterized using nuclear magnetic resonance (NMR) and mass spectroscopy. The micellar and interfacial parameters like the critical micelle concentration (CMC), surface pressure at the CMC (Пcmc), surface tension at CMC (
γcmc), counterion binding (β), maximum surface excess concentration (
Γmax), minimum area per molecule (
Amin), standard free energy of micellization (normalΔGmic0) and adsorption (normalΔGads0), and Kraft temperature were evaluated using surface tension and conductometry methods in aqueous solution as well as in buffer solution. Dynamic light scattering (DLS) was used to determine the size of the micelles formed in the aqueous solution. Cytotoxicity tests were carried out on the C6 glioma cancerous brain cell line using the MTT assay (3‐(4, 5‐dimethylthiazole‐2‐yl)‐2, 5‐diphenyltetrazolium bromide) to evaluate the IC50 value of all the synthesized amphiphiles. Thermal stability of these amphiphiles was also evaluated using a thermal gravimetric analyzer (TGA). Economic way of synthesis, moderate thermal stability, a low value of CMC, and cytotoxicity of these amphiphiles will inspire new research in the field of nanobiotechnology and pharmaceutical industries.