Background: Tugboat crewmembers are susceptible to fatigue during their 24-h work shifts, despite the availability of rest time. The fatigue experienced by seafarers contributes to marine accidents and metabolic and cardiovascular diseases, which have long-term effects. This study aimed to analyse the association between working hours and fatigue and other possibly related factors in tugboat crewmembers. Method: This comparative cross-sectional study included 127 tugboat crew members from 15 randomly chosen tugboats in Samarinda Harbor, Indonesia. Their fatigue levels while at work were measured using a reaction timer and standardised questionnaire. Personal and occupational data of crewmembers, including age, marital status, rating (job ranking), duration on board, length of seafaring experience, watch system, smoking status, coffee and alcohol consumption, and working hours, were collected. Moreover, sleep quality and stress levels related to work-family conflict were measured and analysed using the Pittsburgh Sleep Quality Index (PSQI) and Work-Family Conflict Scale (WCFS), respectively. Results: The study found that 40.2% of the subjects were classified as having fatigue. The determinant factors were long working hours (> 72 h/week), poor sleep quality, and work-family conflict [adj. OR = 13.32; 95%-CI (4.78?31.23)] and p < 0.001, [adj. OR = 4.49 (1.39?14.52)] and p = 0.012, [adj. OR = 2.87 (1.12?7.33)] and p = 0.028, respectively. However, personal and occupational factors, including age, marital status, duration on board, length of seafaring experience, smoking status, and coffee and alcohol consumption, were not significantly associated with crewmember fatigue. Conclusion: The incidence of fatigue among Indonesian tugboat crewmembers operating on the Mahakam River was considerably high. Working hours, sleep quality, and work-family conflict were strongly associated with fatigue in tugboat crewmembers; therefore, the working hours of tugboat crewmembers need to be improved. Crewmember lifestyle variables need to be studied further. ? 2021, The Author(s).