Night work can lead to social jetlag (SJL), which can be briefly defined as the difference between social and biological time. In this sense, SJL has been viewed as a proxy for circadian misalignment. Studies have suggested that SJL may modify physiological processes, such as blood pressure, glucose metabolism, cortisol, and melatonin production. Therefore, we aimed to verify the correlation between SJL and nocturnal inhibition of melatonin production estimated by the concentration of its urinary metabolite (6-sulfatoximelatonin). The study included day workers (n = 9) and night workers (n = 13) from a public maternity hospital in the city of Sao Paulo. A questionnaire was used to obtain sociodemographic data, life habits, working conditions, and the Munich Chronotype Questionnaire (MCTQ(shift)) was used to assess chronotype. Urine was collected on workdays and days off to estimate the concentration of 6-sulfatoximelatonin (aMT6s), quantified by the ELISA method. We found SJL 13 times higher for night workers (10.6 h) than day workers (0.8 h). The excretion of aMT6s in night workers was statistically different on workdays as opposed to days off, with the lowest excretion on workdays, as expected. SJL was correlated with the aMT6s's delta between the night off and night on among night workers, indicating that the higher is the SJL, the lower is the melatonin production. As expected, social jetlag was higher among night workers, compared to day workers. Moreover, our findings showed that melatonin concentration is directly correlated with SJL.