PURPOSE: Although it is well known that the size, shape, and surface chemistry affect the biological potential of silver nanoparticles (AgNPs), the published studies that have considered the influence of AgNP surface on antibacterial activity have not provided conclusive results. This is the first study whose objective was to determine the significance of the surface net charge of AgNPs on their antibacterial potential, attraction to bacterial cells, and cell envelope disruption, considering differences in bacterial surface properties. METHODS: We evaluated five commercial AgNP colloids with identical size and shape but different surface ligands. We thoroughly characterized their physicochemical properties, including the zeta potential, hydrodynamic diameter, and polydispersity index, and determined the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC), along with silver absorption into bacterial cells. Moreover, we investigated structural changes in bacteria treated with AgNPs by using a crystal violet assay and electron microscopy. RESULTS: The zeta potential of AgNPs ranged from -47.6 to +68.5 mV, with a hydrodynamic diameter of 29-87 nm and a polydispersity index of 0.349-0.863. Bacterial susceptibility varied significantly (0.5 ≤ MIC ≤ 256 µg Ag/mL; 1 ≤ MBC ≤ 256 µg Ag/mL); we found the lowest susceptibility in bacteria with a cell wall or a polysaccharide capsule. The most active AgNPs (0.5 ≤ MIC ≤ 32 µg Ag/mL; 2 ≤ MBC ≤ 64 µg Ag/mL) had a moderate surface charge (-21.5 and +14.9 mV). The antibacterial potential was unrelated to ion dissolution or cell envelope disruption, and bacterial cells absorbed less of the most active AgNPs (1.75-7.65%). CONCLUSION: Contrary to previous reports, we found that a moderate surface charge is crucial for the antibacterial activity of AgNPs, and that a significant attraction of the nanoparticle to the cell surface reduces the antibacterial potential of AgNPs. These findings challenge the existing views on AgNP antibacterial mechanisms and interactions with bacterial cells.