Silver nanoparticles were biosynthesized using the culture supernatant of Staphylococcus sp. YRA, a strain isolated from Colombian mining sediments. Synthesis was optimized at 1 mM AgNO₃, pH 7, 40 °C and 7 μg/mL extract, producing spherical, protein-capped AgNPs with primary sizes in the tens-of-nanometers range (~35–90 nm by SEM), while DLS indicated larger hydrodynamic diameters (~250–320 nm) consistent with aggregation in suspension (ζ-potential −16.6 mV). These nanoparticles remained stable over 6 months. Characterization by UV–Vis, SEM, AFM, EDS and FTIR confirmed extracellular protein-mediated reduction and capping. The AgNPs showed antibacterial activity against multidrug-resistant clinical isolates (Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Salmonella bongori, Enterococcus spp.), with inhibition zones of 8–16 mm at 400–1000 μg/mL. Biofilm formation was reduced by >50% at 700 μg/mL in both Gram-positive and Gram-negative strains. In Phaseolus vulgaris (P. vulgaris), low concentrations (5–100 μg/mL) increased growth and chlorophyll content, while 500 μg/mL caused moderate inhibition. FTIR analysis identified amide and thiol groups from bacterial enzymes as capping agents. These results suggest Staphylococcus sp. YRA as a bacterial platform for AgNPs production with antibiofilm activity against MDR pathogens and acceptable phytotoxicity profile for potential applications.