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The growing incidence of human monkeypox cases emphasizes the significance of prevention, early detection, and prompt responses for healthcare providers. The aim of this study was to assess the knowledge and attitudes toward monkeypox infection among physicians, a frontline healthcare worker group, in Saudi Arabia. A cross-sectional survey assessing knowledge and attitudes towards monkeypox infection on multiple-item scales was sent to physicians in Saudi Arabia. The associations between independent factors and either knowledge or attitude were assessed. The final analysis included 398 participants. Approximately 55% of the surveyed participants had a “good knowledge” score about human monkeypox. The adjusted logistic regression analysis showed that being a female physician, working in the private sector, and having information on human monkeypox during medical school or residency years were the only factors associated with a good level of knowledge about human monkeypox. However, physicians’ knowledge and attitudes regarding monkeypox infection are inadequate and influenced by various factors. There is a significant knowledge gap between the therapeutic management of monkeypox and its vaccination. Training and knowledge assessments are important, especially when studies show significant improvement in related and specific knowledge.