In this study, biopolymer-based antimicrobial fibrous membranes constituted by polylactic acid (PLA) and ultrasonically dispersed ZnO nanoparticles (NPs) were developed by electrospinning. The morphology, mechanical properties, thermodynamics and antimicrobial properties of membranes with different ZnO NPs content were comprehensively studied. The results showed that the best performance was achieved when the content of ZnO content was 0.5 wt% (PLA/ZnO-0.5%). Specifically, the tensile strength and elongation at break of the composite membrane were 6.85 ± 0.19 MPa and 74.90 ± 0.59%, respectively, and the inhibition zones of Escherichia coli and Staphylococcus aureus were 15.02 ± 0.18 mm and 14.74 ± 0.06 mm, respectively. In addition, an appropriate ultrasonication time of 45 min improved the thermal, ultraviolet (UV) barrier, and antibacterial properties of the composite membrane. At the 45 min ultrasonication time, the inhibition zone of PLA/ZnO-0.5% against Escherichia coli and Staphylococcus aureus was 18.11 ± 0.15 mm and 15.80 ± 0.11 mm, respectively. Therefore, PLA/ZnO-0.5% can be applied in food packaging to provide an antibacterial effect and UV light barrier.