Air pollution induced by pathogenic particulate matter (PM) has posed a serious threat to public health worldwide. Advanced air filters are thus required, not only exhibiting high PM capture efficiency, low breathing resistance, and high internal moisture transferring performance but also isolating and inactivating external pathogenic aerosols. In this study, we demonstrated a facile approach to construct a biomimetic fibrous leaf-vein membrane with unidirectional water penetration and effective antibacterial PM filtration by one-step electrospinning of poly(vinylidene fluoride) (PVDF)-based multilayer nanofibers. With ultrathin fibers penetrating the skeletal framework of bimodal thick fibers, the membranes showed gradient interconnected porous structures and achieved a highly efficient and stable (in an acid and alkali environment) PM0.3 interception (>99.98%) with low air drag (51-71 Pa). In addition, the gradient narrow pores of the membranes contributed to a gradient higher hydrophilicity. The subsequent unidirectional water motion effectively isolates pathogenic aerosols typically generated by external individuals or ultrafast water penetration from the inverse face. Moreover, the membranes demonstrated an antibacterial efficacy (>99.99%) in a 5 min contact, inactivating the intercepted airborne pathogens efficiently. The test results proved that the proposed membranes were promising advanced air filters for respirator applications.