The melatonin as the pineal gland’s secretory product is implicated in the pathophysiology of migraine. Melatonin has critical functions in human physiology, and research underscores the importance of melatonin in circadian rhythm, sleep, and mood regulation. Clinical observations have indicated that migraine attacks have a seasonal, menstrual, and circadian timing, suggesting that chronobiological mechanisms and their alterations may causally involve in the etiology of the disease. However, the topic has received relatively little attention in the migraine literature. Associations between melatonin, circadian preference, sleep, and mood states were investigated in the current study. Fifty-five patients (47 females and 8 males) were compared to 57 gender and age-matched control subjects (40 females and 17 males). A socio-demographical questionnaire, the Beck Depression Inventory, Beck Anxiety Inventory (BAI), Pittsburgh Sleep Quality Index (PSQI), Profile of Mood States (POMS), and Morningness–Eveningness Questionnaire were administered to volunteers. Blood samples were taken from all participants at about 1:00 AM in an unlit room not to hamper melatonin secretion, and blood melatonin levels were measured using quantitative ELISA test. In comparison with controls, melatonin levels were significantly lower among migraine patients. Migraineurs reported significantly greater scores on the BAI, confusion-bewilderment subscale of the POMS, and total and sleep latency subscale of the PSQI. Migraine patients who had nausea during the migraine attacks and who reported bouts relevant to certain food consumption, such as cheese or chocolate, had significantly lower levels of melatonin. Contrarily, groups did not reveal statistically substantial difference in circadian preferences.