Encapsulation with biodegradable polymers is an alternative to reduce adverse effects and improve the physicochemical properties of metallic nanoparticles. In this study, spherical polymeric nanoparticles with an average size of 200 nm loaded with silver nanoparticles (AgNPs) were obtained. The antimicrobial activity against Escherichia coli O157:H7, methicillin-resistant Staphylococcus aureus (MRSA), and yeasts as Candida albicans, Candida parapsilosis and Candida guilliermondii was determined. MIC90 values of nanocomposites were between 0.01 to 1 μg/mL, potentialized effect up to 500 times compared to free AgNPs. In addition, cytotoxic effect on 50% of murine fibroblast (CC50) was obtained at a mean concentration of 9.57 μg/mL of AgNPs (up to ~1000 times higher than MIC90). Consequently, the polymeric nanocomposites loaded with AgNPs are a potential alternative in the development of new biocide agents on Candida species and pathogenic bacteria at non-toxic concentrations for mammalian cells such as murine fibroblasts.