Electrospun nylon 6,6 composite nanofiber membranes containing TiO2 or SiO2 nanoparticles were fabricated, and their physical and electrochemical properties were assessed for use as high-strength, thermally stable separators for lithium-ion batteries. Experimental results demonstrated that TiO2/nylon 6,6 and SiO2/nylon 6,6 nanofiber membranes not only displayed good mechanical strength and excellent thermal stability, but also showed improved electrochemical properties compared to commercial polypropylene membrane separator. Larger liquid electrolyte uptake, higher ionic conductivity, higher electrochemical oxidation limit, and lower interfacial resistance with lithium were obtained for TiO2/nylon 6,6 and SiO2/nylon 6,6 nanofiber membranes. Among all membranes studied, SiO2/nylon 6,6 nanofiber membranes showed the highest ionic conductivity and lowest interfacial resistance with lithium owing to their highest porosity and well-dispersed nanoparticles. In addition, Li/LiCoO2 and Li/LiFePO4 cells containing these composite nanofiber membranes demonstrated high cell capacities, good cycling performance, and superior C-rate performance at room temperature.