Considering the advantages of electrospun thermoplastic polyurethane (TPU)-fibers and antibiotic (vancomycin)-containing polyvinyl alcohol (PVA)-microbeads for wound dressings, a composite membrane was developed. Electrospun-aligned TPU-fibers were collected using a high-speed drum collector. Three groups of TPU-only, double-sided PVA-microbeads composite TPU-fibers (PVA/TPU/PVA sandwich, DSV40), and simultaneous electrospray PVA-microbeads/electrospun TPU-fibers (interstitial PVA-microbeads, CV40) composite membranes were prepared and characterized. The composites were tested for morphology, infrared spectroscopy, and tensile tests along the fiber-parallel (MD) and fiber-perpendicular (CD) directions, water wettability and absorption, antibacterial activity, and biocompatibility. The results showed that the tensile strength, modulus, and strain of TPU-only in MD and CD directions were 7.31 ± 1.13 and 2.47 ± 0.43 MPa, 2.73 ± 0.36 and 0.69 ± 0.07 MPa and 285.02 ± 23.56 and 360.70 ± 34.01%, respectively. Moreover, MD-direction tensile properties of DSV40 and CV40 resulted in a significant increase in strength (9.05 ± 2.19 and 9.77 ± 1.51 MPa) and modulus (10.97 ± 1.65 and 5.47 ± 0.94 MPa), but a decrease in strain (92.42 ± 11.68 and 185.78 ± 24.85%), compared to TPU-only. However, DSV40 and CV40 showed opposite trends (respective strength of 0.99 ± 0.07 and 2.11 ± 0.33 MPa, modulus of 0.21 ± 0.01 and 0.48 ± 0.51 MPa, and strain of 460.69 ± 29.38 and 435.92 ± 29.26%) in the CD direction compared to TPU-only. In terms of wettability after 10 s observation, DSV40 obtained a hydrophilic tendency (25.91°) than CV40 (74.22°) and TPU-only (121.06°). The results of antibacterial activity showed that both DSV40 and CV40 had good antibacterial effects on Staphylococcus aureus but the inhibitory effect on Escherichia coli was poor. The biocompatibility test confirmed that neither DSV40 nor CV40 negatively affected L929 cell viability.