Many natural organic compounds with pharmaceutical applications, including antibiotics (chlortetracycline and vancomycin), antifungal compounds (pyrrolnitrin), and chemotherapeutics (salinosporamide A and rebeccamycin) are chlorinated. Halogenating enzymes like tryptophan 7-halogenase (PrnA) and tryptophan 5-halogenase (PyrH) perform regioselective halogenation of tryptophan. In this study, the conformational dynamics of two flavin-dependent tryptophan halogenases—PrnA and PyrH—was investigated through molecular dynamics simulations, which are in agreement with the crystallographic and kinetic experimental studies of both enzymes and provide further explanation of the experimental data at an atomistic level of accuracy. They show that the binding sites of the cofactor-flavin adenine dinucleotide and the substrate do not come into close proximity during the simulations, thus supporting an enzymatic mechanism without a direct contact between them. Two catalytically important active site residues, glutamate (E346/E354) and lysine (K79/K75) in PrnA and PyrH, respectively, were found to play a key role in positioning the proposed chlorinating agent, hypochlorous acid. The changes in the regioselectivity between PrnA and PyrH arise as a consequence of differences in the orientation of substrate in its binding site.