Polyelectrolyte multilayer film (PMF) is conventionally fabricated by the layer-by-layer (LBL) assembly of a pair of oppositely charged polyelectrolytes on a substrate through electrostatic attractions. However, the lack of long-term stability of PMF under physiological conditions limits its application as antimicrobial coating in medical devices. In this study, a stable PMF composed of only polyethyleneimine (PEI) was constructed by covalent LBL deposition. First, the specific buildup of PEI during covalent LBL assembly was validated by UV-Vis absorption spectroscopy and atomic force microscopy. Second, silver (Ag) nanoparticles were incorporated into PEI multilayers through in situ reduction of Ag(+) by the pre-absorption of NaBH(4). It was also shown that the mass of Ag nanoparticle can be controlled by varying multilayer thickness and loading cycles. Bacterial live/dead assay showed that the PEI multilayers effectively killed Staphylococcus aureus and Escherichia coli upon contact formation. The inclusion of Ag nanoparticles in (PEI) film not only enhanced the antimicrobial property against adherent bacteria but also led to the inhibition of the bacteria growth in suspended culture via the long-term release of Ag(+) into the liquid medium.