Antibacterial nanomaterials have attracted great interest in recent years, especially with an increase in antibiotic resistance of microbial organisms. However, deleterious properties such as aggregation, toxicity of nanoparticles, and low stability limit their practical application. In this respect, we have developed novel PLA-based fibrous mats with GO-Ag hybrid nanofillers through electrospinning for minimizing bacterial attachment and growth for biomedical applications. Polylactic acid (PLA) exhibits low tensile modulus and strength as well as no bactericidal ability. To enhance its tensile and bactericidal performances, 1 wt % graphene oxide (GO), and 1-7 wt % silver nanoparticle (AgNP) are incorporated into the PLA matrix. For comparison, electrospun PLA-1 wt % GO and PLA-AgNP nanocomposites have also been prepared. The morphological, mechanical and thermal properties as well as bactericidal activities of electrospun PLA-based nanocomposite fibrous mats have been investigated. Tensile tests show that the addition of 1 wt % GO or 1-7 wt % AgNPs to PLA leads to a drastic increase in its elastic modulus. Further enhancements in tensile modulus and strength of PLA can be obtained by adding GO-AgNP nanohybrids. The thermal stability of PLA is greatly improved by adding GO-AgNP nanohybrids. Agar disk diffusion test results indicate that the PLA-1 wt %GO nanocomposite has no inhibition zones against and . However, GO nanofillers with lateral width of micrometer range act as effective anchoring sites for AgNPs. Thus, PLA-1 wt %GO-(1-7) wt % Ag hybrid fibrous mats exhibit excellent antibacterial effect against , while the PLA-1 wt %GO-Ag mats with higher AgNP loadings show bacterial inhibition toward . The bactericidal effects of PLA-1 wt %GO-(1-7)%Ag hybrids are studied and analyzed using live/dead fluorescent imaging assay, quantitative antibacterial efficacy test, SEM examination and residual oxygen species measurement. Our work highlights the development of electrospun nanocomposite mats as promising antibacterial materials for biomedical applications and systematically depicts the bactericidal mechanism of PLA-GO-Ag nanocomposites.