Enzymes play crucial roles in the catalysis of biochemical reactions, facilitating and regulating essential metabolic pathways in living organisms. Given their important functions, enzymes are outstanding targets for the development of inhibitory compounds aimed at disease control. In addition, many pathogenic organisms often rely on enzymes for colonization, survival, and adaptation within the host environment, using them as virulence factors that contribute to disease progression by disrupting the host's immune mechanisms. The discovery of enzyme inhibitors can be based on in silico approaches, such as structure- or ligand-based virtual screening, which allow the identification of potential compounds by predicting interactions between enzymes and candidate molecules stored in specific databases. To evaluate inhibitor efficacy in biological systems and validate computational findings, enzymatic assays involving techniques such as colorimetry and fluorescence can be applied. Thus, this chapter aims to elucidate the main mechanisms of enzyme action and their roles in the survival and virulence of pathogenic organisms, highlighting their relevance in the development of inhibitory compounds targeting both pathogen- and host-derived enzymes for disease control, as well as the key tools employed in this process.