Proteins with a Toll/interleukin-1 receptor/resistance (TIR) domain are among the most ancient immune regulators and include well-known pattern recognition receptors (PRRs). A specialized subset of TIR domain proteins are enzymes that predominantly use nicotinamide adenine dinucleotide (NAD+) to generate second messenger metabolites. These enzymatic TIR proteins have essential roles in bacteria, plant, and animal immunity. The mechanism of activation of these TIR proteins, conserved across Kingdoms, involves oligomerization into higher-ordered structures, which activates their intrinsic enzymatic activity. Here, we review the functions of enzymatic TIR proteins in innate immunity in bacteria, plants, and animals. This work offers insights into the evolutionary origins of immunity itself and defines fundamental principles of immune surveillance across the Tree of Life.