The natural hydrophilic property of leather makes it a possible growing substrate for microorganisms. Microorganisms such as bacteria, yeasts, and molds frequently grow on shoe lining leather, causing skin diseases, unpleasant odors, and discomfort for the wearers. To address these issues, herein, bio-synthesized silver nanoparticles from Piper betle L. leaf extract were applied as an antimicrobial agent to pig leather via the exhaustion method. The characterization of nanosilver-treated pig leather was performed using colorimetry, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), atomic absorption spectroscopy (AAS), and FTIR techniques. Evaluation of antimicrobial efficacy of the nanosilver-treated leather was both qualitatively and quantitatively assessed against two bacteria (Escherichia coli and Staphylococcus aureus) and two fungi (a yeast Candida albicans and a mold Aspergillus niger) in accordance with AATCC TM90, AATCC TM30, and ISO 16187:2013 standards. The results indicated that the nanosilver was immobilized on the surface of collagen fibers as well as within the collagen matrix of the pig leather. The treated leather exhibited highly effective antibacterial and antifungal activities against all tested microorganisms, and the inhibition increased with an increase in the initial nanosilver concentration in the treated solution. Furthermore, the exhaustion technique used for the antimicrobial treatment of pig leather had no negative effects on its physico-mechanical properties, and it met the standard requirements of ISO 20882:2007 for shoe upper lining. Therefore, based on the efficient antimicrobial and suitable physico-mechanical properties, nanosilver-treated pig leather adapts the criteria for making hygienic shoe upper lining.