Lactoperoxidase (LPO) found in saliva, tears and airways protects system against bacterial and viral attack. It utilized hydrogen peroxide in the presence of halides or pseudohalide forming hypohalous acid a potent biocidal and virocidal agent. Melatonin is an important biomolecule that controls a number of functions including circadian sleep rhythms, blood pressure, oncogenesis, retinal function, seasonal reproduction, and immunity. Additionally, melatonin is frequently used as a supplement in a variety of medical conditions such as jet lag, shift work, and circadian rhythm sleep disorders, cancer, longevity and antioxidant therapy. Here, we demonstrate that melatonin modulates classic catalytic mechanism of LPO under physiological-like conditions. In the presence of chloride (SCN-), melatonin inactivated LPO classic peroxidase cycle by formation of compound II or two different intermediate compounds that are dead end products of LPO system. This behavior indicates that melatonin modulates the formation of LPO intermediates and their decay rates to the ground state this efficiently slowing down or inhibiting the cycling of the enzyme. Importantly, melatonin-dependent inhibition of LPO depends on hydrogen peroxide concentrations. Thus, the interplay between LPO and melatonin can have a broader implication especially in the cases of supplemental melatonin therapy.