Free-standing polyelectrolyte multilayer films which are composed of biomacromolecules, such as polysaccharides and/or polypeptides, have attracted numerous attentions in biomedical fields. However, the poor mechanical strength makes their future applications difficult. In the present work, we used the pH-amplified Poly-(l-lysine)/Hyaluronic acid (PLL/HA) film as the template and in situ synthesized silver nanoparticles (AgNPs) in a green way. It was confirmed that the size of AgNPs in the films was largely dependent on the loading method of Ag+ (pre-complex vs. post-loading). Meanwhile, the loading amount of AgNPs and their distribution in the films could be controlled by the films outermost layer (PLL vs. HA) as observed under electron microscopies. The reduced AgNPs were used here as both bactericidal agent and mechanical reinforcer. The anti-bacterial activity of the films with AgNPs was verified by the Kirby-Bauer assay, and film cytotoxicity was examined by MTT examination. The effects of AgNPs' amount, size and distribution on the mechanical properties of films were evaluated by both nanoindentation and micro tensile tests. The films embedded with AgNPs of smaller size and well-distribution throughout the films gained the maximal enhancement on mechanical strength and preserved acceptable cytocompatibility. The in situ UV reduction of silver nanoparticles in polyelectrolyte multilayer films with controllable internal structures could provide a facile way to ensure the films with enhanced mechanical properties as well as effective anti-bacterial and normal-cell-supporting capabilities.