This study aimed to develop a safe and advanced antibacterial material of electrospun microfiber membranes (MFMs) for wound dressings. Combinations of several materials were investigated; thermal treatment and electrospinning techniques were used to form the best quality of MFMs to suit its end applications. By comparing the fiber morphology, diameter changes, and fracture strength, the suitable ratio of raw materials and thermal treatment were obtained before and after adding Trition X-100 as a surfactant for MFMs of sodium alginate/polyvinyl alcohol/polyethylene oxide (SA/PVA/PEO). The electrospinning solution was mixed with berberine as an antibacterial substance; meanwhile, calcium chloride (CaCl2) was used as the crosslinking agent. The antibacterial properties, water dissolution resistance, water content, and fracture strength were thoroughly investigated. The results showed that the antibacterial rates of MFMs with different mass fractions of berberine (0, 3, and 5 wt.%) to Escherichia coli (E. coli) were 14.7, 92.9, and 97.2%, respectively. The moisture content and fracture strength of MFMs containing 5 wt.% berberine were 72.0% and 7.8 MPa, respectively. In addition, the produced MFMs embodied great water dissolution resistance. Berberine-loaded SA/PVA/PEO MFMs could potentially serve as an antibacterial wound dressing substrate with low cost and small side effects.