Generation of microbicidal reactive oxygen species (ROS) is a pivotal protective component of the innate immune system in many eukaryotes. NOD (nucleotide oligomerisation domain containing protein)-like receptors (NLRs) have been implicated as phylogenetically ancient sensors of intracellular pathogens or endogenous danger signals. NOD2 recognizes the bacterial cell wall component muramyldipeptide leading to NFκB and MAPK activation via induced proximity signalling through the serine-threonine kinase RIP2. In addition to the subsequent induction of cytokines and antimicrobial peptides, NOD2 has been shown also to exert a direct antibacterial effect. Using a fluorescence-based ROS detection assay we demonstrate controlled ROS generation as an integral component of NOD2-induced signalling in epithelial cells. We demonstrate that the NAD(P)H oxidase family member DUOX2 is involved in NOD2-dependent ROS production. Coimmunoprecipitation and fluorescence microscopy were used to show that DUOX2 interacts and colocalizes with NOD2 at the plasma membrane. Moreover, simultaneous overexpression of NOD2 and DUOX2 was found to result in cooperative protection against bacterial cytoinvasion using the Listeria monocytogenes infection model. RNAi-based studies revealed that DUOX2 is required for the direct bactericidal properties of NOD2. Our results demonstrate a new role of ROS as effector molecules of protective cellular signalling in response to a defined danger signal carried out by a mammalian intracellular NLR system.