ABSTRACT: Background: The incidence rate of coronavirus disease 2019 (COVID-19) is increasing in several countries despite that public health measures are put in place. Given that COVID-19 is a newly emerging disease, there is little knowledge about the disease. The present study aims to assess knowledge, perception, and preventive practices toward COVID-19 among health workers in Tigray, North Ethiopia. Materials and Methods: A health facility-based cross-sectional study was conducted among health professionals working in public hospitals. Data were collected between April and May 2020. The researchers included 403 participants and recruited them via a simple random sampling technique. To collect data, the researchers prepared a structured questionnaire guided by the WHO survey questions. Data were entered into Epi-info 7 and exported to SPSS version 20.00 for analysis. The researchers applied descriptive and inferential statistical analyses. Tables and graphs were used to describe data, and multivariate binary logistic regression was used to determine factors affecting knowledge, perception, and practices toward COVID-19 prevention. Results: Among the participants, 79, 88, and 64.3% of them had adequate knowledge, positive perception, and good practice toward preventing COVID-19, respectively. Besides, 92% of the study participants knew that the COVID-19 virus does not have curative treatment and vaccine. The findings revealed that 55% of the respondents did not use the necessary personal protective equipment (PPE) at all times. The result showed that being female [AOR: 2.43, 95% CI (1.50-3.94)] and having a work experience of 2-5 years [AOR: 2.44, 95% CI (1.10-5.39)], news media as a source information [AOR: 7.11, 95% CI (3.07-16.49)], social media as a source information [AOR: 4.59, 95% CI (2.15-9.84)], and governmental website as a source information [AOR: 4.21, 95% CI (2.15-8.27)] were reported as protective factors; and being single [AOR: 0.15, 95% CI (0.03-0.75)] was reported as risk factor toward the prevention of COVID-19. Conclusion: Most health workers had adequate knowledge and positive attitude toward COVID-19; nevertheless, a significant proportion of health workers had poor practice toward the prevention of COVID-19, including the use of PPE. Additionally, some groups of health professional showed poor practices of implementing the public health measures, hence the call for them to improve in the prevention and control of COVID-19.