Nature is an incredible source of inspiration for the discovery and subsequent development of new bioactive compounds. Unfortunately, the synthesis of these molecules is often prohibitively complex, requiring the installation of multiple functional groups with intricate three-dimensional architectures critical to their biological activity. Biocatalytic reactions embody many features of ideal chemical transformations, including the potential for impeccable selectivity, high catalytic efficiency, mild reaction conditions, and the use of environmentally benign reagents. These advantages have created a demand for new biocatalysts that expand the portfolio of complexity-generating reactions available to synthetic chemists. Oxidative dearomatization is a powerful transformation in the synthesis of complex molecules, as it generates a stereocenter and simultaneously producing a compound primed for further reactions. Nature has developed a class of biocatalysts, flavin-dependent monooxygenases, which perform this reaction with an excellent site- and stereoselectivity under mild conditions. Our studies on the TropB-catalyzed hydroxylation of phenolic compounds has defined the substrate scope of these biocatalysts; however, the mechanistic underpinnings were a mystery. Through analysis of class A FAD monooxygenases and biochemical characterization of TropB we determined that the phenolate form of the substrate interacts with Tyr239 and Arg206 to control the site- and stereo-selectivity of the hydroxylation. We then we explore how this control for site- and stereo-selective is translated to a selection of FAD-dependent monooxygenases. Through a sequence-profiling approach, we identified the FDMO AfoD with complementary selectivity compared to TropB. We determined by probing similarly positioned residues through mutagenesis and biochemical characterization that selectivity can be eroded when Tyr118 hydrogen bonding is affected. These findings pave the way in identifying new biocatalysts for reaction development toward natural product synthesis.