The kinetics of mercuric ion (Hg2+) binding with heterogeneous naturally dissolved organic matter (DOM) has been hypothesized to result from competitive interactions among different organic ligands and functional groups of DOM for Hg2+. However, an experimental protocol is lacking to determine Hg2+ binding with various competitive ligands and DOM, their binding strengths, and their dynamic exchange reactions. In this study, a stepwise reduction approach using ascorbic acid (AA) and stannous tin [Sn(II)] was devised to differentiate Hg(II) species in the presence of two major functional groups in DOM: the carboxylate-bound Hg(II) is reducible by both AA and Sn(II), whereas the thiolate-bound Hg(II) is reducible only by Sn(II). Using this operational approach, the relative binding strength of Hg2+ with selected organic ligands was found in the order dimercaptopropanesulfonate (DMPS) > glutathione (GSH) > penicillamine (PEN) > cysteine (CYS) > ethylenediaminetetraacetate > citrate, acetate, and glycine at the ligand-to-Hg molar ratio < 2. Dynamic, competitive ligand exchanges for Hg2+ from weak carboxylate to strong thiolate functional groups were observed among these ligands and within DOM, and the reaction depended on the relative binding strength and abundance of thiols and carboxylates, as well as reaction time. These results provide additional insights into dynamic exchange reactions of Hg2+ within multicompositional DOM in controlling the transformation and bioavailability of Hg(II) in natural aquatic environments.