Hypothesis: Modification of the interface by blending of surfactants produces considerable changes in the elastic rigidity of the interface, which in turn affects the physicochemical properties of w/o microemulsions. Hence, it could be possible to tune the thermodynamic properties, microstructures and antimicrobial activity of microemulsions by using ionic/non-ionic mixed surfactants and polar lipophilic oil, which are widely used in biologically relevant systems. Experiments: The present report was aimed at precise characterization of mixed cetyltrimethylammonium bromide and polyoxyethylene (23) lauryl ether microemulsions stabilized in 1-pentanol (Pn) and isopropyl myristate at different physicochemical conditions by employing phase studies, the dilution method, conductivity, DLS, FTIR (with HOD probing) and H-1 NMR measurements. Further, microbiological activities at different compositions were examined against two bacterial strains Bacillus subtilis and Escherichia coli at 303 K. Findings: The formation of mixed surfactant microemulsions was found to be spontaneous at all compositions, whereas it was endothermic at equimolar composition. FTIR and H-1 NMR measurements showed the existence of bulk-like, bound and trapped water molecules in confined environments. Interestingly, composition dependence of both highest and lowest inhibitory effects was observed against the bacterial strains, whereas similar features in spontaneity of microemulsion formation were also evidenced. These results suggested a close relationship between thermodynamic stability and antimicrobial activities. Such studies on polar lipophilic oil derived mixed surfactant microemulsions have not been reported earlier. (C) 2014 Elsevier Inc. All rights reserved.