Electrospun SiO2/nylon 6,6 nanofiber membranes were fabricated and their electrochemical performance was evaluated for use as separators in Li-ion batteries. The aim of this study was to design new high-performance separator membranes with enhanced mechanical properties and good thermal stability, as well as superior electrochemical performance compared to microporous polyolefin membranes. It was found that SiO2/nylon 6,6 nanofiber membranes had superior thermal stability and mechanical strength with highly porous structure. Enhanced electrochemical properties were also obtained for these nanofiber membranes due to their high porosity values. Compared with commercial microporous polyolefin membranes, SiO2/nylon 6,6 nanofiber membranes had larger liquid electrolyte uptake, higher electrochemical oxidation limit, and lower interfacial resistance with lithium. SiO2/nylon 6,6 nanofiber membranes with different SiO2 contents (0, 3, 6, 9 and 12%) were assembled into lithium/lithium cobalt oxide and lithium/lithium iron phosphate cells. High cell capacities and good cycling performance were demonstrated at room temperature. In addition, cells using SiO2/nylon 6,6 nanofiber membrane separators showed superior C-rate performance compared to those using commercial microporous polyolefin membrane.