With the continuous application of antibacterial materials, various problems have emerged, such as expensive prices and the potential development of resistance. Cationic antibacterial agents, due to their high solubility, reactivity, and antibacterial properties, are considered as environmentally friendly and cost-effective antibacterial agents. In addition, the electrospinning technique is recognized as a versatile and high-efficiency method to produce nanofibers with multifunctional properties and adjustable structures. In this work, we prepared a series of nanofiber membranes by electrospinning technology using hexadecyl trimethyl ammonium Bromide (CTAB) and 5-Chloro-8-hydroxyquinoline (5-Cl8Q) as antibacterial agents and polylactic acid (PLA) as substrate. The antimicrobial performance of PLA/CTAB/5-Cl8Q was the highest among the prepared materials, which inhabited S. aureus and E. coli up to 99.9% and 95.9%, respectively, and the antibacterial properties were stable. In general, PLA/CTAB/5-Cl8Q has great development potential, and it can be applied to real life as a cost-effective, biodegradable and highly antibacterial material.