In this work, we synthesize dodecyl mercaptan-functionalized silver nanoparticles integrated with polypropylene nanocomposite (DM-AgNPs/PP) substrates by a simple in situ melt blending method. The formation and distribution of AgNPs are confirmed by UV-visible spectroscopy, Fourier transform infrared spectroscopy, transmission electron microscopy, and thermogravimetric analysis. The existence of DM-AgNPs in PP film substrate enhances the thermal degradation and crystallization properties. Further, the antimicrobial activity of as-synthesized DM-AgNPs/PP film substrate is studied using Gram-negative ( Escherichia coli) and Gram-positive ( Staphylococcus aureus) bacteria as model microbes, which displayed significantly enhanced bacteriostatic activities under optimized composition and experimental conditions. Interestingly, PP substrate with 0.4% DM-AgNPs exhibits drastically improved antibacterial property via the release of oxygen reactive species and Ag ion diffusion processes; thus, the inhibition rates of E. coli and S. aureus are obtained as 100 and 84.6%, respectively, which is higher than the conventional AgNPs. Finally, we demonstrate the migration study of Ag ions from the DM-AgNPs/PP film using different food simulant solutions by inductively coupled plasma-mass spectrometry analysis and the dissolved Ag ion content is estimated, which is a key prospect for the toxicity analysis. The overall Ag ion migration value is estimated between 1.8 and 24.5 μg/cm2 and displayed a lowest limit of Ag ion migration as 0.36 μg/cm2. Our work highlights the development of high performance nanocomposites as promising antibacterial and food simulant materials for biomedical and industrial applications.