Microorganisms can attack and kill nematodes by diverse processes such as capturing, parasitizing, and producing toxins and enzymes. Extracellular enzymes, including serine proteases, chitinases, and collagenases are shown to be important virulence factors that can degrade the main chemical constituents of the nematode cuticle and eggshell. Here, we review the structure, function, regulation, and evolution of these nematicidal enzymes and provide insights into the mechanisms of microbial infections against nematodes. We discuss the practical applications of these nematicidal enzymes in agriculture and other areas.