In this study, ZnO-loaded PA6 nanocomposite preparation and its antibacterial activity are investigated. This work aims to study the effect of the sizes and amount of the ZnO nanofiller on the antibacterial, mechanical, and thermal properties of the PA6/ZnO nanocomposites. The melt intercalation method is applied to prepare polyamide 6 (PA6) nanocomposite fibers, including 0, 0.5, 1, 3, 5 wt % zinc oxide (ZnO), using a laboratory-scale compounder. The antibacterial activity of the fibers against Staphylococcus aureus (ATCC 6538) as a gram positive bacterium and Klebsiella pneumoniae (ATCC 4352) as a gram negative bacterium is determined according to ASTM E 2149-0. Mechanical and thermal characterization tests are performed according to relevant standards (ASTM D7426-08, ASTM E1131-08, ASTM D3822-07; DSC, TGA, tensile tests). It is found that the dispersion of the ZnO particles within the PA6 matrix is homogenous according to scanning electron microscopy results. Antibacterial activity tests show that PA6/ZnO nanocomposite fibers exhibit antibacterial efficiency related to their nanoparticle contents. An increase in the amount of nanoparticles causes an increase of the antibacterial activity of the fibers. On the other hand, mechanical and thermal characterization tests show that the addition of ZnO nanoparticles does not affect the strength and thermal properties of the nanocomposites for these loadings.