Background The burden of communicable diseases within developing countries is mainly influenced by poor personal hygiene practices. Hand washing is considered as most cost effective intervention for reducing health problems such as diarrhoea and acute respiratory tract infections. This study aimed to assess hand washing practice at critical times and identify associated factors among mothers of under five children in Debark town. Method A community based cross-sectional study design was carried out from May 1–20, 2018 in Debark town. After selection of participants using simple random sampling, face to face interview was performed by using semi-structured pre-tested questionnaire. Data were entered into EPI Info 7 and exported into SPSS 21 for further analysis. Results were presented by simple frequency, percentage and mean for descriptive variables. Binary logistic regression analysis was used to test the association of dependent and independent factors. Variables with 95% confidence interval and p ≤ 0.2 during the univariable binary logistic regression analysis were included in the multivariate logistic regression analysis. At the final model variables with p ≤ 0.05 were treated as significantly associated factors of hand washing practice at critical times. Results Good hand washing practice at critical times was reported in 52.2% (95% CI: 47.5, 57.2%) of study participants. Desirable attitude [AOR = 3.37, 95% CI (2.03, 5.58)], presence of water for washing hands [AOR = 4.86, 95% CI (1.26, 18.69)] and a good knowledge [AOR = 2.98, 95% CI (1.92, 4.60)] were significantly associated factors with hand washing practice at critical times. Conclusion The hand washing practice at critical times of study participants was found to be low. A significant proportion of mothers of under five children have a poor hand washing practice at critical times. It is necessary to increase the access to water and to improve knowledge and attitude of mothers to improve their hand washing practice at critical times.