We report here the synthesis of uniform nanospheres-like silver nanoparticles (AgNPs, 5-10 nm) and the dumbbell-like Fe3O4-Ag hybrid nanoparticles (FeAgNPs, 8-16 nm) by the use of seeding growth method in the presence of oleic acid (OA)/oleylamine (OLA) as surfactants. The antibacterial activity of pure nanoparticles and nanocomposites by monitoring the bacterial lag–log growth has been investigated. The electron transfer from AgNPs to Fe3O4NPs which enhances the biological of silver nanoparticles has been proven by nanoscale Raman spectroscopy. The lamellae structure in the spherulite of FeAgNPs/PE nanocomposites seems play the key role to the antibacterial activity of nanocomposites, which has been proven by nanoscale AFM-IR. An atomic force microscopy coupled with nanoscale infrared microscopy (AFM-IR) is use to highlight the distribution of nanoparticles on the surface of nanocomposite at the nanoscale. The presence of FeAgNPs in PE nanocomposites has a better antibacterial activity than that reinforced by AgNPs due to the faster Ag+ release rate from the Fe3O4-Ag hybrid nanoparticles and the ionization of AgNPs in hybrid nanostructure.