In this work different variables have been analyzed in order to optimize the bactericidal properties of chitosan films loaded with silver nanoparticles. The goal was to achieve complete elimination of antibiotic resistant and biofilm forming strains of Staphylococcus aureus after short contact times. The films were produced by solution casting using chitosan as both a stabilizing and reducing agent for the in situ synthesis of embedded silver nanoparticles. We have applied an innovative approach: the influence of the chitosan molecular weight and its deacetylation degree (DD) were analyzed together with the influence of the bacterial concentration and contact time. The best results were obtained with high DD chitosan where a fast reduction was favored; leading to smaller nanoparticles (nucleation is promoted), and a sufficiently high polymer viscosity prevented the resulting nanoparticles from undesired agglomeration. In addition, for the first time, potential detachment of the silver nanoparticles from the films was evaluated and neglected, demonstrating that uncontrolled release of silver nanoparticles from the chitosan films is prevented. The influence of the ionic silver released from the films, silver loading, nanoparticle sizes, contact, and initial number of bacteria was also analyzed to elucidate the mechanism responsible for the strong bactericidal action observed.