Among metal-based nanoparticles, silver nanoparticles (AgNPs) are particularly appealing because of their stability, functionality, and documented antimicrobial properties. AgNPs also offer the possibility of different surface modifications. In this work, we functionalized AgNPs with thiobarbituric acid or 11-mercaptoundecanoic acid residues to improve the nanoparticles’ biological activities. Subsequently, we assessed the physicochemical properties of newly synthesized AgNPs using a wide range of biophysical methodologies, including UV/vis and fluorescence spectroscopy, atomic force and scanning electron microscopy, and dynamic light scattering and isothermal titration calorimetry. Next, we examined the effect of nanoparticles functionalization on AgNPs mutagenicity and toxicity. Our study revealed that AgNPs’ surface modification affects nanoparticles aggregation, and also impacts nanoparticles’ interaction with model acridine mutagen ICR-191. AgNPs coated with MUA showed the most interesting interactions with tested ICR-191, slightly modulating its toxicity properties by decreasing the viability in treated cells.