This work focuses on the synthesis, characterization and assessment of biological behavior of hydrophilic silver nanoparticles (AgNPs) for innovative applications in the field of nanobiotechnology. AgNPs functionalized with two capping agents, i.e., the 3-mercapto-1-propansulfonate (3MPS) and 1-β-thio-D-glucose (TG), were on purpose synthesized and extensively characterized. The investigation of the structural and electronic properties of the complex nanosystems was carried out by means of X-ray Photoelectron Spectroscopy (XPS), with the aim to probe the nature of the interactions at the metal/organic ligands interface as well as the stability of the functionalized systems and capping molecules. XPS data allowed us to achieve a deep understanding of the influence of the thiols stoichiometric ratio on the electronic properties and stability of AgNP-3MPS-TG, as well as to ascertain the efficiency of TG loading. Moreover, in order to shed lights on the local properties at Ag atoms sites of the nanostructure bulk, X-Ray Absorption Spectroscopy was also applied. Preliminary studies about the antibacterial properties of our nanostructured materials were then performed in bacterial strains of Bacterial pathogens belonging to the high-antibiotic resistant ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter sp.), providing promising results for applications in the field of clinical therapy against high-antibiotic resistant bacteria.