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Zewdu Bishaw Aynalem,1 Melsew Dagne Abate,1 Fentahun Meseret,2 Ahmed Nuru Muhamed,3 Gebremeskel Kibret Abebe,4 Abebaw Bires Adal,1 Adam Wondmieneh,1 Atsedemariam Andualem,1 Sewunet Ademe,1 Haile Workye,1 Bekalu Bewket,1 Getachew Amare Beyene,5 Tamiru Alene,6 Tilahun Degu Tsega7 1Department of Nursing, College of Medicine and Health Sciences, Injibara University, Injibara, Ethiopia; 2Department of Pediatrics and Child Health Nursing, School of Nursing and Midwifery, College of Health and Medical Science, Haramaya University, Harar, Ethiopia; 3Department of Nursing, College of Medicine and Health Science, Wolkite University, Wolkite, Ethiopia; 4Department of Emergency and Critical Care Nursing, School of Nursing, College of Health Sciences, Woldia University, Woldia, Ethiopia; 5Department of Midwifery, College of Medicine and Health Sciences, Injibara University, Injibara, Ethiopia; 6Department of Pediatrics and Child Health Nursing, College of Medicine and Health Sciences, Injibara University, Injibara, Ethiopia; 7Department of Public Health, College of Medicine and Health Sciences, Injibara University, Injibara, EthiopiaCorrespondence: Zewdu Bishaw Aynalem, Department of Nursing, College of Medicine and Health Sciences, Injibara University, Injibara, Ethiopia, Tel +251918375722, Email Zewdu.bishaw@inu.edu.et; zest7@yahoo.comBackground: Fighting health threats, especially the rise of new infectious diseases, is one of the main responsibilities of healthcare workers. However, their knowledge and attitudes toward monkeypox have not yet been assessed. Therefore, this study aimed to assess the knowledge, attitude, and factors associated with monkeypox infection among healthcare workers at Injibara General Hospital, Northwest Ethiopia.Methods: Institution-based cross-sectional study was conducted from December 1– 30, 2022. Data were collected using a structured self-administered questionnaire. A simple random sampling technique was used to select study participants. Descriptive statistics and multivariable logistic regression analyses were computed. The degree of association was interpreted using an odds ratio with a 95% confidence interval and a p-value < 0.05.Results: Among the 200 healthcare workers who participated, 38.5% (95% CI: 32.5%– 45%) and 62% (95% CI: 55– 68.5%) had good knowledge and positive attitudes regarding monkeypox respectively. Having a master’s degree or above (AOR = 11.25: 95% CI: 2.03– 62.33), being vaccinated against COVID-19 (AOR = 2.60: 95% CI: 1.37– 4.94), and having access to information about monkeypox (AOR = 3.37: 95% CI: 1.33– 8.50) were the factors associated with good knowledge. Furthermore, a positive attitude was significantly associated with being 30 years of age or older (AOR = 2.95: 95% CI: 1.55– 5.60) and having access to information about monkeypox (AOR = 4.14: 95% CI: 2.06, 8.30).Conclusion: Both good knowledge and positive attitudes were relatively low among healthcare workers. Factors such as age, education level, COVID-19 vaccination status, and access to information about monkeypox were significantly associated with the knowledge and attitude of healthcare workers. To enhance the knowledge and attitude of healthcare workers, hospitals should consider offering educational upgrades, hosting educational events like seminars, conferences, webinars, and campaigns, and ensuring comprehensive coverage of the topic in medical curricula.Keywords: monkeypox, mpox, knowledge, attitude, associated factors, healthcare worker, Ethiopia, Injibara