Lithium-Sulfur batteries (LSBs) are one of the most promising next-generation batteries to replace Li-ion batteries that power everything from small portable devices to large electric vehicles. LSBs boast a nearly five times higher theoretical capacity than Li-ion batteries due to sulfur’s high theoretical capacity, and LSBs use abundant sulfur instead of rare metals as their cathodes. In order to make LSBs commercially viable, an LSB’s separator must permit fast Li-ion diffusion while suppressing the migration of soluble lithium polysulfides (LiPSs). Polyolefin separators (commonly used in Li-ion batteries) fail to block LiPSs, have low thermal stability, poor mechanical strength, and weak electrolyte affinity. Novel nanofiber (NF) separators address the aforementioned shortcomings of polyolefin separators with intrinsically superior properties. Moreover, NF separators can easily be produced in large volumes, fine-tuned via facile electrospinning techniques, and modified with various additives. This review discusses the design principles and performance of LSBs with exemplary NF separators. The benefits of using various polymers and the effects of different polymer modifications are analyzed. We also discuss the conversion of polymer NFs into carbon NFs (CNFs) and their effects on rate capability and thermal stability. Finally, common and promising modifiers for NF separators, including carbon, metal oxide, and metal-organic framework (MOF), are examined. We highlight the underlying properties of the composite NF separators that enhance the capacity, cyclability, and resilience of LSBs.