Silver nanoparticles exert high antibacterial activity and thus have been used in polymeric formulations for drug delivery. In recent years, polymeric formulations containing silver nanoparticles have been found to be highly effective against biofilm infections, which are difficult to treat with drugs only. However, the antibacterial effects of silver nanoparticles in polymer composites depending on administration time are still unknown. In this study, we used transmission electron microscopy to investigate the antibacterial activity of silver decorated poly(dl-lactide--glycolide) (PLGA) (Ag PLGA) nanoparticles against biofilms at different treatment times. LIVE/DEAD assay result showed that approximately 85% of the bacteria in the biofilms was killed after 6 h of administering the Ag PLGA nanoparticles. The formulation comprising Ag PLGA nanoparticles was found to be highly effective and to exhibit low cytotoxicity. However, silver nanoparticles were ejected from the bacterial cells up to 4 h after treatment administration due to the self-protection properties of the bacteria. On the basis of the results, we propose a potential mechanism for the antibacterial activity of silver nanoparticles per treatment time, taking into account the detoxification activity of bacterial cells. This information can contribute not only to an understanding of foreign-body elimination but also to the design of effective formulations against biofilm infections.