Biocatalysis, the application of enzymes as catalysts for chemical synthesis, has become an increasingly valuable tool for the synthetic chemist. Enzymatic transformations carried out by enzymes or whole-cell catalysts are used for the production of a wide variety of compounds ranging from bulk to fine chemicals. The primary consideration for the incorporation of biotransformation in a synthetic sequence is regio- and stereocontrol that can be achieved with enzyme-catalyzed reactions. Biotransformations are thus becoming accepted as a method for generating optically pure compounds as well as for developing efficient routes to target compounds. This Special Issue aims to address the main applications of biocatalysts, isolated enzymes, and whole microorganisms in the synthesis of bioactive compounds and their precursors.