Aim: Melatonin (MT) is a neurohormone produced and secreted primarily by the pineal gland in a circadian manner, and mainly acts through 2 receptor subtypes: MT1 and MT2 in humans. The diversity in their tissue distribution is in favor of different functions for each receptor subtype. Selective modulators are therefore required to determine the physiological roles of these melatonin receptor subtypes and their implications in pathological processes. Methods: A homogenous MT1/MT2 receptor binding assay was established for high-throughput screening of new ligands at the hMT1 and/or hMT2 receptors. The functional properties (agonists or antagonists) were assessed by a conventional guanosine-5′[γ-35S] triphosphate (GTP-γS) assay. Results: Three hMT1 receptor-selective small molecule antagonists and 1 hMT2 receptor-selective small molecule antagonist with novel structural features were identified following a high-throughput screening campaign of 48 240 synthetic and natural compounds. Conclusion: The findings may assist in the expansion of chemical probes to these 2 receptor subtypes.