BACKGROUND Obesity is associated with increased general mortality and comorbidities, it is multifactorial and some evidence has shown that sleep duration and shift work may be implicated in its pathogenesis. OBJECTIVE The aim of this study was to evaluate the association between shift work, quality of life and obesity among healthcare workers of a Brazilian University Hospital. METHODS A cross-sectional study was performed from April 2013 to December 2014 with 200 workers of a University Hospital. Sociodemographic data were evaluated and BREF WHOQOL was used for quality of life. The physical activity was evaluated using the International Physical Activity Questionnaire (IPAQ), Chronotypes and daily sleep preference were investigated using Munich Chronotype Questionnaire (MCTQ). Venous blood was collected after 12-h of fasting for laboratory tests. RESULTS In this sample, the night shift workers had higher income and were older compared to day shift workers. Night shift workers sleep less hours, had higher weight, body mass index and abdominal circumference when compared to the day shift workers. Night shift workers had almost 3 times higher association with abdominal obesity independent of age and gender, than day shift workers. MCTQ parameters showed that night shift workers had lower sleep duration during working days and also during free days, associated with a higher level of social jetlag. Social jetlag had an association with obesity. We found no difference for quality of life between shifts. CONCLUSIONS Night work was a risk factor for abdominal obesity, social jetlag was higher in night shift workers and it was associated with presence of obesity.