Clostridium difficile (C. difficile) infections (CDIs) are a leading cause of hospital-acquired infections, often associated with high recurrence rates due to multidrug resistance. This study explores a novel formulation of Fidaxomicin-loaded silver nanoparticles (Fid-AgNPs) as an advanced antimicrobial strategy against C. difficile. The nanoparticles were synthesized using a green synthesis method and characterized for size, morphology, and surface properties. Dynamic Light Scattering (DLS) analysis confirmed the nanoparticle size to be 85 ± 5 nm with a polydispersity index (PDI) of 0.22, indicating uniform size distribution. The zeta potential measured at -28.5 mV confirmed high stability. Drug loading efficiency (DLE) was optimized at 78.3%, and the release kinetics showed a sustained drug release of 85% over 72 hours. In-vitro antibacterial assays demonstrated enhanced efficacy of Fid- AgNPs compared to free Fidaxomicin. The Minimum Inhibitory Concentration (MIC) of Fid-AgNPs was found to be 0.5 µg/mL, significantly lower than the MIC of free Fidaxomicin (1.5 µg/mL). Zone of inhibition (ZOI) tests revealed an average zone diameter of 22.5 ± 1.8 mm for Fid-AgNPs, indicating strong antibacterial activity against multidrug-resistant C. difficile strains. Biofilm disruption assays showed a 65% reduction in biofilm formation, highlighting the formulation’s potential to prevent recurrent infections. The synergistic effect of Fidaxomicin and silver nanoparticles resulted in a 2-fold increase in antibacterial activity, as evidenced by the fractional inhibitory concentration index (FICI) value of 0.4. These findings suggest that Fid-AgNPs offer a promising, effective, and synergistic strategy against drug-resistant C. difficile infections, with potential applications in reducing recurrence rates and improving patient outcomes.