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Background. Exclusive breastfeeding (EBF) practices are the infant only receives breast milk for the first 6 months of life. This is expected to be well known and practised by healthcare workers who are in the position to educate, promote and support the community. Objective. To analyze factors that affect on exclusive breastfeeding practices by healthcare workers at the Semarang District Hospital. Methods. The research was observational analytic with cross-sectional design, the samples were healthcare workers at the Semarang District Hospital who had 6-24 month-old babies. The data were gathered by using questionnaires and analyzed by using chi-square test and logistic regression analysis. Results. Ninety eight healthcare workers were recruited, including doctors, nurses, pharmacists and other healthcare workers. Only 43,9 % healthcare workers practiced EBF. The main reason why they did not practiced EBF was being a busy work schedule. Knowledge (OR CI95%=3.028 p=0.046) and availability of facilities (OR CI95%=2.849 p=0.036) were related to exclusive breastfeeding practice. Conclusion. Because of shortcomings in exclusive breastfeeding practices among the healthcare workers, they need to be empowered and supported to promote and support exclusive breastfeeding. To improve their exclusive breastfeeding practices and knowledge, fulfill the facilities and training periodically should be conducted.