Introduction: Infections associated with medical implants pose significant challenges to healthcare, demanding the development of new effective antimicrobial strategies. This study focuses on the creation of a novel biological membrane with enhanced antimicrobial properties by with the incorporation of either vancomycin or silver nanoparticles. Methods: Pericardium samples were impregnated with either silver nanoparticles (AgNPs) or vancomycin (VAN). The antimicrobial activity of the membranes was assessed through qualitative microbiological analysis, biofilm production and animal in vivo model of infection. Histological analysis was performed on the pericardium samples, and quantitative microbiological analysis was conducted on the tissue from the animal model and biocompatibility evaluation. Statistical analyses were carried out using appropriate tests. Results: Microbiological tests confirmed both VAN and AgNP showed inhibition halos against bacteria. Histological analysis indicated minimal inflammatory response and bacterial presence in VAN and AgNP groups compared to the control group revealed reduced bacterial presence. Biomechanical testing showed that polypropylene had the lowest tensile strength, while AgNP-impregnated pericardium exhibited lower resistance than VAN-impregnated and control pericardium. Conclusion: Lyophilized and decellularized bovine pericardium as a substrate, combined with the incorporation of vancomycin or silver nanoparticles, provides a versatile platform for combating infections associated with medical implants.