The relationship between the prevalence of multiple sclerosis (MS) and sunlight's ultraviolet radiation was proved. Oxidative stress plays a role in the pathogenic traits of MS. Melatonin possesses antioxidative properties and regulates circadian rhythms. Sleep disturbances in MS patients are common and contribute to daytime fatigue. The aim of study was to evaluate 5 mg daily melatonin supplementation over 90 days on serum total oxidant status (TOS), total antioxidant capacity (TAC) and its influence on sleep quality and depression level of MS patients. A case-control prospective study was performed on 102 MS patients and 20 controls matched for age and sex. The Kurtzke's Expanded Disability Status Scale, magnetic resonance imaging examinations, Athens Insomnia Scale (AIS), Beck Depression Inventory questionnaires were completed. Serum TOS and TAC levels were measured. We observed higher serum levels of TOS in all MS groups, while after melatonin treatment the TOS levels significantly decreased. The TAC level was significantly lower only in mitoxantrone-treated group and it increased after melatonin supplementation. A strong positive correlation between T1Gd(+) number lesions and TAC level in interferon-beta-1A group was observed. AIS group mean score above 6 defining insomnia were observed in interferon-beta-1B-group, glatiramer acetate-group and mitoxantrone-group: 6.62 ± 2.88, 8.45 ± 2.07, 11.1 ± 3.25, respectively. After melatonin treatment the AIS mean scores decrease in glatiramer acetate-group and mitoxantrone-group achieving 5.25 ± 1.14 and 7.08 ± 2.39, respectively (p < 0.05). Finding from our study suggest that melatonin can act as an antioxidant and improves reduced sleep quality in MS patients.