Sutures are a crucial component of surgical procedures, serving to close and stabilize wound margins to promote healing. However, microbial contamination of sutures can increase the risk of surgical site infections (SSI) due to colonization by pathogens. This study aimed to tackle SSI by synthesizing positively charged silver nanoparticles (P-AgNPs) and using them to produce antimicrobial sutures. The P-AgNPs were reduced and stabilized using polyethylenimine (PEI), a cationic branched polymer. The physiochemical characteristics of P-AgNPs were confirmed from the surface plasmon resonance (SPR) peak at 419 nm, spherical morphology with a particle size range of 8−10 nm, PEI functional groups on NPs, a hydrodynamic diameter of 12.3 ± 2.4 nm, and a zeta potential of 31.3 ± 6 mV. Subsequently, the surfaces of silk sutures were impregnated with P-AgNPs at different time intervals (24, 48, and 96 h) using an ex situ method. Scanning electron microscopy (SEM) and tensile strength studies were conducted to determine the coating and durability of the NP-coated sutures. The NPs were quantified on sutures using inductively coupled plasma optical emission spectrophotometry (ICP-OES), which was in the range of 1−5 μg. Primarily, antimicrobial activity was studied using three microorganisms (Candida albicans, Streptococcus mutans, and Staphylococcus aureus) for both P-AgNPs and suture-coated P-AgNPs using the agar diffusion method. The results showed that only the NPs and NP-coated sutures exhibited enhanced antimicrobial effects against bacteria and fungi. Finally, the cytotoxicity of the sutures was investigated using stem cells from the apical papilla (SCAPs) for 24 h, which exhibited more than 75% cell viability. Overall, the results indicate that NP-coated sutures can potentially be used as antimicrobial sutures to diminish or inhibit SSI in postoperative or general surgery patients.