The evolution of antibiotics-resistant bacteria is considered a major concern. To explore promising antibacterial materials and clarify their unknown mechanisms, the mode of action of silver nanoparticles (AgNPs) against Salmonella enterica serotype typhimurium was investigated. We investigated the effect of AgNPs on the bacterial membrane. The N-phenyl-1-naphthylamine assay showed that the permeability of the outer membrane was not changed by treatment with AgNPs. The O-nitrophenyl-beta-d-galactopyranoside assay showed that the inner membrane permeability increased as AgNPs concentration increased. Our results showed that AgNPs affected the inner membrane without outer membrane damage. Generally, antibiotic-induced reactive oxygen species (ROS) and changes in the Ca2+ gradient are known to contribute to bacterial cell death. Likewise, we detected that AgNPs induced the accumulation of ROS and intracellular Ca2+ depending on its concentration, using 2',7'-dichlorodihydrofluorescein and Fura-2AM, respectively. At higher concentrations, no relationship between oxidative stress and bactericidal effects of AgNPs was confirmed through a cell viability assay and intracellular Ca2+ assay with antioxidant N-acetylcysteine. In this study, the inner membrane disruption followed by membrane dysfunction played a key role in the antibacterial activity of AgNPs against S. typhimurium. Contrary to the expected results, ROS do not influence growth inhibition of AgNPs.