Objective: to analyse the differences between genders in the description in the professional, domestic and total work hours and assess its association with health-related behaviour among nurses. Methods: this is a transversal study carried out in 18 different public hospitals in the municipality of Rio de Janeiro. The data collection procedure was based on questionnaires. All nurses working with assistance were considered eligible (n=2,279). Results: men and women showed significant differences in relation to working hours. The female group showed longer domestic and total work hours when compared to the group of men. In contrast, the number of hours spent on professional work was higher among men. For the women, both the professional hours and total work hours were often associated with excessive consumption of fried food and also coffee, lack of physical exercise and also the greater occurrence of overweight and obesity. Conclusion: both the professional hours and the domestic work hours need to be taken into account in studies about health, self-care and also the care provided within the context of nursing workers, particularly among women. The results add weight to the need for actions for health promotion in this occupational group and the importance of assessing the impact of long working hours on the health of workers.