Control of microbial infections is one of the growing concerns in modern societies due to the increasing presence and risk of pathogenic bacterial spread. Therefore, there is a strong demand to design new antibacterial agents to overcome these problems. The aim of this study is the preparation of a new series of biocompatible starch-based polyurethane (PU) nanocomposites with a unique combination of magnetic nanoparticles (MNPs) and silver nanoparticles (AgNPs). In order to perform the study, first, a new synthetic diisocyanate was synthesized by a three-step reaction and grafted into bis(2-hydroxyethyl terephthalate) (BHET) and starch as a covering agent of MNPs containing different amounts of AgNPs. Afterward, the diisocyanate containing two active isocyanate moieties was reacted with hydroxyl groups of BHET and starch to form stable urethane linkages. Subsequently, the antibacterial activity of the pure synthetic PU and the produced nanocomposites were investigated against Escherichia coli and Staphylococcus aureus as Gram-negative and Gram-positive microorganism models, respectively. The obtained results exhibited that the nanocomposites have a synergistic effect on the antibacterial activity, which decreased with increasing of the Ag content. Such results may be related to the AgNPs agglomeration in the nanocomposite