Proper shipping of infectious materials is an important practice to prevent healthcare workers (HCWs), other people and environment from spread of biological threats. The main purpose of this study was to determine healthcare providers' knowledge, and practices about transportation of infectious substances. A self-administered designed questionnaire was used to collect relevant data from study respondents. Primary data were double entered and analyzed by using statistical package for social sciences (IBM SPSS statistics 20). Descriptive, in addition to the effects of predictors on dependent variables were applied. Multiple logistic regression model was used to assess the association between dependent variables and predictors. It has been found that; the overall mean percentage of knowledge was 57.1 (95 CI: 54.1, 60.2), while mean percentage of practice was 68.9 (CI: 95% 66.7 -71.1). Response reliability scale was 89.0% based on Cronbach's alpha. The highest knowledge regarding transportation of infectious substances was observed among laboratory specialist (33.9%) and doctors (25.4%) respectively. High percentage of practice was observed among laboratory specialist was (34.6%) and low percentage was among public health officers and doctors (15.9%). Majority of study respondents did not receive training in transportation of infectious substances (70.9%). Knowledge and practice on transportation of infectious substances among health care providers are low. More efforts are needed to fill the gap in knowledge and practice by applying effective training programs that can lead to increase skills and awareness of health care providers.