The resistance of pathogenic bacteria to antibiotics has become a serious problem. The emphasis is placed on the development of new, effective antimicrobial strategies. One of them is the use of AgNPs in association with antibiotic drugs. The aim of this study was to obtain silver nanoparticles functionalized with ampicillin and to investigate the mechanism of binding antibiotics to nanoparticle using high-performance liquid chromatography approach. To confirm the occurrence of silver nanoparticles functionalization, FTIR, MALDI-TOF MS, and DLS analysis and zeta potential measurements were performed. Moreover we assessed the antibacterial activity of biologically synthesized nanoparticles functionalized with ampicillin against a range of gram (+) and gram (-) bacteria strains such as Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus epidermidis, and Escherichia coli.