Nanofibers are one of the most attractive materials that can modify functionalities for developing new kinds of specific applications, mainly used as a biomedical material. Herein, we designed and prepared the antibacterial nonwoven fiber mats of PLA and PLA composited with Ag nanoparticles by electrospinning. The effects of varying filler contents on their chemical, surface morphology, thermal, water absorbency, and antibacterial properties were investigated using FTIR, SEM/EDS, DSC, swelling ratio, and qualitative and quantitative antibacterial tests. FTIR and EDS spectra indicated that Ag nanoparticles were incorporated in the PLA without chemical bonding. SEM revealed that the average diameter of the PLA nanofibers containing the Ag nanoparticles was more significant than those without those particles. Besides, fiber diameters are proportionally the amount of Ag nanoparticle contents. DSC indicated that the Ag nanoparticles can be incorporated within the PLA-matrix without strongly affecting their thermal properties. Moreover, the crystallinity of the composite nonwoven fiber mats was higher than those of fiber mats in the neat PLA. Accordingly, the antibacterial activities revealed that all the composite nanofiber mats exhibited excellent against S. aureus and E. coli bacterial strains. In addition, in the cell toxicity study, all produced hybrids nonwoven fiber mats induced a reduction in cell viability for the L929 fibroblast cells. Our results suggest that the designed and prepared nonwoven fiber mats may have good potential for use in the biomedical field, particularly in wound dressing applications