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Background: Monkeypox outbreaks in non-endemic countries emphasize the importance of being prepared to prevent its progression to a pandemic. To effectively control monkeypox, healthcare providers must have sufficient knowledge and good attitudes and practices to limit its spread. We initiated this project to assess the factors associated with health workers' knowledge and attitude toward monkeypox in southwestern Saudi Arabia. Methods: We included 398 eligible health workers working at various health facilities. Data was collected using an online survey, and participants had an opportunity to consent. We conducted descriptive statistics for all variables and used chi-square statistics, t-test, and multivariate analysis to establish the association between health workers' demographic characteristics and knowledge of monkeypox disease. Results: The mean age was 30.93 ± 8.25 years for the included participants, and most of them were between 22 and 29 years, male, single, nurses, working in government hospitals, and had worked for at least five years. The chi-square and t-test showed that the participants' knowledge level was significantly related to age, marital status, job title, and medical practice. Most of the participants had low knowledge and good attitudes toward monkeypox prevention measures. Multivariate analysis showed that higher knowledge was associated with younger age after controlling all other significant bivariate relationships between knowledge and demographics. Conclusions: This study found low knowledge levels and high good attitude levels of monkeypox among the participants. As such, there is a need to support health workers in understanding monkeypox epidemiology, prevention, and treatment. Therefore, Saudi Arabia will be making significant strides to being well prepared and ready to handle future monkeypox outbreaks. [ABSTRACT FROM AUTHOR]