Antimicrobial materials have attracted much attention all over the world. Herein, a new kind of antimicrobial material, poly (ether amide) (Pebax) nanofibers containing Ag nanoparticles, was prepared by electrospinning method. The Ag/Pebax composites were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), differential scanning calorimetry (DSC), and thermogravimetric analyzer (TGA) measurements. The antimicrobial properties of Ag/Pebax composites against Escherichia coli (E. coli; ATCC25922 and avirulent) and Staphylococcus aureus (S. aureus; ATCC6538 and avirulent) were evaluated by membrane adhering method. It was found that the Ag content played an important part in the antimicrobial ability of Ag/Pebax composites. When the mass ratio of AgNO3 to Pebax in the precursor was 0.15%, the inhibition rate can reach >99.9 parts per thousand and antimicrobial activity against E. coli and S. aureus was 5.8 and 5.6, respectively, exceeding the antimicrobial testing standards JIS Z 2801. The above results indicated that the Ag/Pebax composite was a promising antimicrobial material that can be used in many applications.