There is consistent evidence suggesting a relationship between individuals' sleep-wake rhythms and well-being. The indiscriminate demands from daily working routines, which do not respect this individual physiological rhythm, might be mediating this phenomenon. The aim of the present study was to evaluate the relationship between the characteristics of sleep routines during working days and psychological well-being. This was a cross-sectional study on 825 individuals from rural communities from southern Brazil. The study protocol included a questionnaire on demographic characteristics, working routines, health complaints, and habits; the Munich Chronotype Questionnaire for sleep-wake rhythm and; the WHO-Five well-being index. Since sex has been shown to affect sleep circadian rhythm and well-being, analysis was performed on men and women separately. In the proposed hierarchical regression models, different factors contributed to well-being according to sex. Among men, sleep-wake and work-related variables did not predict well-being scores. Among women, later midpoints of sleep on working days (B = -1.243, SE B = 0.315, β = -0.220), working more days per week (B = -1.507, SE B = 0.494, β = 0.150), having longer working journeys (B = -0.293, SE B = 0.105, β = -0.166), earlier working journey midpoints (B = 0.465, SE B = 0.222, β = 0.115), and being exposed to less sunlight (B = 0.140, SE B = 0.064, β = 0.103) predicted worse well-being. For the subgroup of women with days free from work, we have found a correlation between later midpoints of sleep during the week with worse well-being (Pearson's r = -0.159, p = 0.045) while the same relationship was not significantly observed with the midpoint of sleep on non-working days (Pearson's r = -0.153, p = 0.054). Considering WHO-Five as categorical, based on proposed clinical cut-offs, among women working 7-d/week, those with worst well-being (WHO-Five < 13) had the latest midpoint of sleep (F = 4.514, p = 0.012). Thus, the midpoint of sleep on working days represents the interaction between individuals' sleep-wake behavior and working routines. It plays an important role as a stress factor and may be a useful alternative variable related to chronodisruption.