The pathogenicity of intracellular bacteria was not acquired during interaction with their mammalian hosts, but as a consequence of the interaction with ancient phagocytic protozoan. The selection pressure for bacteria to escape protozoan predation equipped them with specific mechanisms to manipulate the hostile environment of phagosomes. The NADPH oxidase 2 is dedicated to rapid generation of intraphagosomal reactive oxygen species. Our phylogenetic study revealed that NOX enzymes evolved from prokaryotic Ferric Reductases in early eukaryotes. The evolution of NOX enzymes from iron reduction to ROS generation indicates that NOX enzymes became specialized to interactive cell-cell relations and host-pathogen co-evolution. We used the social amoeba Dictyostelium discoideum to study the conserved biological functions of NOX enzymes. We generated single and multiple NOX KOs to study their functions during bacteria infection and killing. Our studies provide further insights into the multifunctional roles of NOX enzymes in a primitive innate immunity model system.