Although silver is known for its antibacterial activity, its exact mode of action remains unclear. In our previous work, we described AgNbO
3
nanoparticles (AgNbO
3
NPs) prepared using a ceramic method, followed by high-energy and low-energy ball-milling processes, which exhibited antimicrobial activity with negligible release of Ag
+
in deionized water. Here, we investigated thoroughly the mode of action of these AgNbO
3
NPs against
Escherichia coli
. Drastic morphological changes in
E. coli
were observed after their exposure to AgNbO
3
NPs. In addition to cellular damage, AgNbO
3
NPs induced the production of reactive oxygen species and lipid peroxidation, likely following the release of small amounts of Ag
+
. This was concluded from the characterization of mutants resistant to AgNbO
3
NPs that showed cross-resistance to AgNO
3
, impaired reactive oxygen species production and lipid peroxidation, and harbored a key mutation in a two-component regulatory system regulating an Ag
+
efflux pump. We calculated, however, that the quantity of Ag
+
released from AgNbO
3
NPs is not sufficient by itself to lead to bacterial death. We propose that bacterial contact with the AgNbO
3
NPs in combination with Ag
+
release is necessary for the mode of action of AgNbO
3
NPs.
IMPORTANCE
Silver is known for its antibacterial activity, but its exact mode of action remains unclear. Here, we investigated thoroughly the mode of action of AgNbO
3
nanoparticles against
Escherichia coli
. Our data suggest that AgNbO
3
nanoparticles have dual effects on the cell and that both are required for its lethal action.