Excessive wound exudates often cause infection and hinder wound repair and regeneration. It is therefore urgently needed to develop an ideal wound dressing for pumping the excessive biofluids and inhibiting bacterial infection in the wound healing process. Herein, we demonstrate a facile and efficient strategy to fabricate the unidirectional water-transport antibacterial wound dressings by embedding macromolecular antimicrobials into a multilayer nanofibrous membrane featured with hydrophobic-to-hydrophilic gradient structure and self-pumping effect using an electrospinning technique. Thanks to these features, the obtained nanofibrous dressings achieved excellent unidirectional water-transport performance, which could drive wound exudates to flow spontaneously from inside to outside and prevent pumped biofluids rewetting the wounds. In addition, the designed trilayered dressing exhibited approximately 100% antibacterial ability against Staphylococcus aureus and Escherichia coli when the concentration of antibacterial agents polyhexamethylene guanidine hydrochloride (PHGC) was 0.06 wt%. Moreover, the trilayered dressings revealed excellent water absorption performance, air and moisture permeability, mechanical strength, biocompatibility, and low cell adhesion behavior, indicating the potential applications for the trilayered nanofibrous wound dressings in wound care.