The effect of exogenous melatonin on ethanol fermentation ability, major fermentation products, antioxidant system, and total antioxidant ability of Saccharomyces cerevisiae was investigated; in addition, the melatonin content in S. cerevisiae was determined. The results showed that fermentation ended on day 8 in the control group, while it continued until day 14 in the treatment groups; however, the total CO2 weight loss, residual sugar content, and alcohol content did not differ significantly between the control and melatonin treatment groups at the end of fermentation. There was a significant decrease in the production of trehalose and glycerol in the treatment groups (after day 8) compared to that observed in the control. The glycerol content in the groups treated with low, medium, and high concentrations of melatonin was observed to be lower by 7.57%, 12.29%, and 11.99%, respectively, compared to that in the control group, which attained a maximum level of 6.25 g/L on day 8. Low concentration melatonin treatment stimulated the activity of superoxide dismutase (SOD) and catalase (CAT) enzymes, and reduced the accumulation of malondialdehyde (MDA) during fermentation. The activity of the SOD was increased in the 0.01 and 1 mg/L melatonin treatment groups by 68.15% and 59.89%, respectively, compared to that in the control; conversely, the CAT activity in the low, medium, and high melatonin treatment groups was observed to increase by 2.85-, 2.87-, and 1.84-fold, respectively, compared to that in the control at the end of fermentation. MDA content in the high melatonin treatment group was 71.44% that of the control (at the end of fermentation). There was an increase in the antioxidant activity of the broth treated with melatonin corresponding to the increase in melatonin concentration. Melatonin concentration gradually decreased during fermentation to reach a steady state level towards the end of the process.