Due to the potential use of bile salts in drug delivery, the mixed micellar, microstrutural and mixed monolayer investigations of three biodegradable diester bearing cleavable gemini surfactants (m-E2-m; m = 12, 14, 16) with sodium deoxycholate and sodium cholate in aqueous media have been carried out by tensiometry, dynamic light scattering and spectrofluorimetry studies. The micellar and adsorption characteristics like composition, mutual interaction, hydrodynamic radius, aggregation number, activity coefficient, minimum area per molecule, and free energies of micellization and adsorption have been evaluated and compared. A synergistic interaction was observed both in the micelle as well as at interface, as evident from interaction parameters. The results are discussed in terms of the structural characteristics and nature of spacer in the gemini surfactants as well as in terms of the presence of hydroxyl groups in bile salts. Furthermore, the m-E2-m geminis generate stronger synergistic interactions with sodium cholate as compared to sodium deoxycholate. Dynamic light scattering (DLS) and spectrofluorimetry results indicate microstructural evolution in the aqueous mixtures of m-E2-m geminis and bile salts which offer potential pharmaceutical applications.