Because of the wide applications of the antibacterial fabrics, they have had broad-based development recently. Chitosan is an environment-friendly, biodegradable and natural organic antibacterial agent, which can be used to prepare the antibacterial fibers. Chitosan-composited electrospun fibers have the potential to be applied in the fields such as surgical gowns, medical gauze, underwear, and sportswear. Therefore, using a one-step 12-needle electrospinning method with liquid bath circulating system, PA6/CS-NPs nanofiber filaments would be fabricated. Under the combined effect of the drafting and impact forces, the surface of the nanofibers could adhere to a certain number of chitosan nanoparticles (CS-NPs). By varying the mass ratios of 0.2 wt.% Span-80/Tween-80 (S-80/T-80) in the bath solution, water flow rate, and the winding rate, the filaments with different properties could be prepared. The results revealed that the surface of PA6 nanofibers could adhere to the largest number of CS-NPs and the mechanical properties were good when the mass ratio of S-80/T-80 was 3/7, the water flow rate was 20 ml/min, and the roller rate was 40 r/min. The nanofibers fabricated by the optimum spinning parameters showed excellent antibacterial activity. In addition, the rate of bacteriostasis against E. coli and S. aureus was 99.9% and 98.9%, respectively.