Background: Exclusive breastfeeding is the most widely known and effective intervention for preventing early-childhood deaths. Optimum breastfeeding practices can prevent 1.4 million deaths worldwide among children under five every year. The aim of this study was to assess the prevalence of exclusive breastfeeding and associated factors among mothers who have an infant less than six months old in Debre Markos, Northwest Ethiopia. Methods: A community based cross-sectional study was conducted from April 1 to 30, 2013. A simple random sampling technique was used from a list of all mothers who had an infant less than six months old obtained from the health extension workers (HEWs) registration book in all kebeles (neighbourhoods) of the city. A total of 423 mothers with infants less than six months old were included in this study. Data were collected using questionnaires administered at interview. Both bivariate and multivariate logistic regression analyses were carried out to identify factors associated with exclusive breastfeeding. Results: The prevalence of exclusive breastfeeding during the seven days before the survey was 60.8% (95% CI: 55.8%, 65.8%). Those mothers who were unemployed [AOR = 1.98 (1.21, 3.22)], received breastfeeding counseling during antenatal care (ANC) [AOR = 2.44 (1.53, 3.91)], received infant feeding counseling during postnatal care (PNC) [AOR = 5.03 (3.04, 8.31)], didn’t give prelacteal feeding [AOR = 3.44 (1.88, 6.33)] and had adequate knowledge about breastfeeding [AOR = 2.57 (1.57, 4.19)] were more likely to practice EBF than their counterparts. Conclusions: Although the prevalence of exclusive breastfeeding was lower in the study area than international recommendations, rates were higher than found in other studies. Recommendations for improving exclusive breastfeeding include better support for working mothers through extending maternal leave and establishing work-site day care centers for infants, expanding the urban health extension program so that more pregnant women and mothers can be taught about appropriate infant and young child feeding practices and how to express their milk, thereby increasing their breastfeeding knowledge.